Liquid formulations for treatment of diseases or conditions

ABSTRACT

Described herein are liquid formulations which deliver a variety of therapeutic agents, including but not limited to rapamycin, to a subject for an extended period of time; liquid formulations which form a non-dispersed mass when placed in an aqueous medium of a subject; liquid formulations comprising a therapeutic agent and a plurality of polymers; and methods for delivering therapeutic agents to a subject for an extended period of time using the liquid formulations. The liquid formulation may be placed in an aqueous medium of a subject, including but not limited to via intraocular or periocular administration. A method may be used to administer rapamycin to treat or prevent angiogenesis, choroidal neovascularization, or age-related macular degeneration in a subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/183,649, filed Jun. 15, 2016, which is a continuation of U.S.application Ser. No. 14/553,947, filed Nov. 25, 2014, now U.S. Pat. No.9,381,153, which is a continuation of U.S. application Ser. No.13/741,103, filed Jan. 14, 2013, now U.S. Pat. No. 8,927,005, which is acontinuation of U.S. application Ser. No. 12/778,872, filed May 12,2010, now U.S. Pat. No. 8,367,097, which is a continuation of U.S.application Ser. No. 11/351,761, filed Feb. 9, 2006, now abandoned,which is related to and claims priority from U.S. ProvisionalApplication No. 60/664,040, filed Mar. 21, 2005, U.S. ProvisionalApplication No. 60/664,306, filed Mar. 21, 2005, and U.S. ProvisionalApplication No. 60/651,790, filed Feb. 9, 2005, each of which isincorporated herein by reference in its entirety for all purposes.

FIELD

Described herein are liquid formulations for treatment, prevention,inhibition, delaying onset of, or causing regression of a disease orcondition by delivery of therapeutic agents to a subject, including butnot limited to a human subject, including but not limited to thetreatment of age-related macular degeneration (“AMD”) by delivery of aliquid formulation comprising a therapeutic agent, including but notlimited to rapamycin (sirolimus), to the eye of a subject, including butnot limited to a human subject. Nonlimiting examples of liquidformulations include solutions, suspensions, and in situ gellingformulations.

BACKGROUND

The retina of the eye contains the cones and rods that detect light. Inthe center of the retina is the macula lutea, which is about ⅓ to ½ cmin diameter. The macula provides detailed vision, particularly in thecenter (the fovea), because the cones are higher in density. Bloodvessels, ganglion cells, inner nuclear layer and cells, and theplexiform layers are all displaced to one side (rather than restingabove the cones), thereby allowing light a more direct path to thecones.

Under the retina are the choroid, comprising a collection of bloodvessels embedded within a fibrous tissue, and the deeply pigmentedepithelium, which overlays the choroid layer. The choroidal bloodvessels provide nutrition to the retina (particularly its visual cells).

There are a variety of retinal disorders for which there is currently notreatment or for which the current treatment is not optimal. Retinaldisorders such as uveitis (an inflammation of the uveal tract: iris,ciliary body, and choroid), central retinal vein occlusive diseases(CRVO), branch retinal venous occlusion (BRVO), macular degeneration,macular edema, proliferative diabetic retinopathy, and retinaldetachment generally are all retinal disorders that are difficult totreat with conventional therapies.

Age-related macular degeneration (AMD) is the major cause of severevisual loss in the United States for individuals over the age of 60. AMDoccurs in either an atrophic or less commonly an exudative form. Theatrophic form of AMD is also called “dry AMD,” and the exudative form ofAMD is also called “wet AMD.”

In exudative AMD, blood vessels grow from the choriocapillaris throughdefects in Bruch's membrane, and in some cases the underlying retinalpigment epithelium. Organization of serous or hemorrhagic exudatesescaping from these vessels results in fibrovascular scarring of themacular region with attendant degeneration of the neuroretina,detachment and tears of the retinal pigment epithelium, vitreoushemorrhage and permanent loss of central vision. This process isresponsible for more than 80% of cases of significant visual loss insubjects with AMD. Current or forthcoming treatments include laserphotocoagulation, photodynamic therapy, treatment with VEGF antibodyfragments, treatment with pegylated aptamers, and treatment with certainsmall molecule agents.

Several studies have recently described the use of laserphotocoagulation in the treatment of initial or recurrent neovascularlesions associated with AMD (Macular Photocoagulation Study Groups(1991) in Arch. Ophthal. 109:1220; Arch. Ophthal. 109:1232; Arch.Ophthal. 109:1242). Unfortunately, AMD subjects with subfoveal lesionssubjected to laser treatment experienced a rather precipitous reductionin visual acuity (mean 3 lines) at 3 months follow-up. Moreover, at twoyears post-treatment treated eyes had only marginally better visualacuity than their untreated counterparts (means of 20/320 and 20/400,respectively). Another drawback of the procedure is that vision aftersurgery is immediately worse.

Photodynamic therapy (PDT) is a form of phototherapy, a termencompassing all treatments that use light to produce a beneficialreaction in a subject. Optimally, PDT destroys unwanted tissue whilesparing normal tissue. Typically, a compound called a photosensitizer isadministered to the subject. Usually, the photosensitizer alone haslittle or no effect on the subject. When light, often from a laser, isdirected onto a tissue containing the photosensitizer, thephotosensitizer is activated and begins destroying targeted tissue.Because the light provided to the subject is confined to a particularlytargeted area, PDT can be used to selectively target abnormal tissue,thus sparing surrounding healthy tissue. PDT is currently used to treatretinal diseases such as AMD. PDT is currently the mainstay of treatmentfor subfoveal choroidal neovascularization in subjects with AMD(Photodynamic Therapy for Subfoveal Choroidal Neovascularization in AgeRelated Macular Degeneration with Verteporfin (TAP Study Group) ArchOphthalmol. 1999 117:1329-1345.

Choroidal neovascularization (CNV) has proven to be recalcitrant totreatment in most cases. Conventional laser treatment can ablate CNV andhelp to preserve vision in selected cases not involving the center ofthe retina, but this is limited to only about 10% of the cases.Unfortunately, even with successful conventional laser photocoagulation,the neovascularization recurs in about 50-70% of eyes (50% over 3 yearsand >60% at 5 years). (Macular Photocoagulation Study Group, Arch.Ophthalmol. 204:694-701 (1986)). In addition, many subjects who developCNV are not good candidates for laser therapy because the CNV is toolarge for laser treatment, or the location cannot be determined so thatthe physician cannot accurately aim the laser. Photodynamic therapy,although utilized in up to 50% of new cases of subfoveal CNV has onlymarginal benefits over natural history, and generally delays progressionof visual loss rather than improving vision which is already decreasedsecondary to the subfoveal lesion. PDT is neither preventive ordefinitive. Several PDT treatments are usually required per subject andadditionally, certain subtypes of CNV fare less well than others.

Thus, there remains a long-felt need for methods, compositions, andformulations that may be used to optimally prevent or significantlyinhibit choroidal neovascularization and to prevent and treat wet AMD.

In addition to AMD, choroidal neovascularization is associated with suchretinal disorders as presumed ocular histoplasmosis syndrome, myopicdegeneration, angioid streaks, idiopathic central serouschorioretinopathy, inflammatory conditions of the retina and or choroid,and ocular trauma. Angiogenic damage associated with neovascularizationoccurs in a wide range of disorders including diabetic retinopathy,venous occlusions, sickle cell retinopathy, retinopathy of prematurity,retinal detachment, ocular ischemia and trauma.

Uveitis is another retinal disorder that has proven difficult to treatusing existing therapies. Uveitis is a general term that indicates aninflammation of any component of the uveal tract. The uveal tract of theeye consists of the iris, ciliary body, and choroid Inflammation of theoverlying retina, called retinitis, or of the optic nerve, called opticneuritis, may occur with or without accompanying uveitis.

Uveitis is most commonly classified anatomically as anterior,intermediate, posterior, or diffuse. Posterior uveitis signifies any ofa number of forms of retinitis, choroiditis, or optic neuritis. Diffuseuveitis implies inflammation involving all parts of the eye, includinganterior, intermediate, and posterior structures.

The symptoms and signs of uveitis may be subtle, and vary considerablydepending on the site and severity of the inflammation. Regardingposterior uveitis, the most common symptoms include the presence offloaters and decreased vision. Cells in the vitreous humor, white oryellow-white lesions in the retina and/or underlying choroid, exudativeretinal detachments, retinal vasculitis, and optic nerve edema may alsobe present in a subject suffering from posterior uveitis.

Ocular complications of uveitis may produce profound and irreversibleloss of vision, especially when unrecognized or treated improperly. Themost frequent complications of posterior uveitis include retinaldetachment; neovascularization of the retina, optic nerve, or iris; andcystoid macular edema.

Macular edema (ME) can occur if the swelling, leaking, and hard exudatesnoted in background diabetic retinopathy (BDR) occur within the macula,the central 5% of the retina most critical to vision. Backgrounddiabetic retinopathy (BDR) typically consists of retinal microaneurismsthat result from changes in the retinal microcirculation. Thesemicroaneurisms are usually the earliest visible change in retinopathyseen on exam with an ophthalmoscope as scattered red spots in the retinawhere tiny, weakened blood vessels have ballooned out. The ocularfindings in background diabetic retinopathy progress to cotton woolspots, intraretinal hemorrhages, leakage of fluid from the retinalcapillaries, and retinal exudates. The increased vascular permeabilityis also related to elevated levels of local growth factors such asvascular endothelial growth factor. The macula is rich in cones, thenerve endings that detect color and upon which daytime vision depends.When increased retinal capillary permeability effects the macula,blurring occurs in the middle or just to the side of the central visualfield, rather like looking through cellophane. Visual loss may progressover a period of months, and can be very annoying because of theinability to focus clearly. ME is a common cause of severe visualimpairment.

There have been many attempts to treat CNV and its related diseases andconditions, as well as other conditions such as macular edema andchronic inflammation, with pharmaceuticals. For example, use ofrapamycin to inhibit CNV and wet AMD has been described in U.S.application Ser. No. 10/665,203, which is incorporated herein byreference in its entirety. The use of rapamycin to treat inflammatorydiseases of the eye has been described in U.S. Pat. No. 5,387,589,titled Method of Treating Ocular Inflammation, with inventor PrassadKulkarni, assigned to University of Louisville Research Foundation, thecontents of which is incorporated herein in its entirety.

Particularly for chronic diseases, including those described herein,there is a great need for long acting methods for delivering therapeuticagents to the eye, such as to the posterior segment to treat CNV in suchdiseases as AMD, macular edema, proliferative retinopathies, and chronicinflammation. Formulations with extended delivery of therapeutic agentare more comfortable and convenient for a subject, due to a diminishedfrequency of ocular injections of the therapeutic agent.

Direct delivery of therapeutic agents to the eye rather than systemicadministration may be advantageous because the therapeutic agentconcentration at the site of action is increased relative to thetherapeutic agent concentration in a subject's circulatory system.Additionally, therapeutic agents may have undesirable side effects whendelivered systemically to treat posterior segment disease. Thus,localized drug delivery may promote efficacy while decreasing sideeffects and systemic toxicity.

SUMMARY

The methods, compositions, and liquid formulations described hereinallow delivery of a therapeutic agent to a subject, including but notlimited to a human subject or to the eye of a subject. Described hereinare methods, compositions, and liquid formulations for delivering avariety of therapeutic agents for extended periods of time which can beused for the treatment, prevention, inhibition, delaying onset of, orcausing regression of a number of conditions or diseases, including butnot limited to diseases or conditions of the eye. The liquidformulations include, without limitation, solutions, suspensions, and insitu gelling formulations.

Described herein are methods, compositions and liquid formulations foradministering to a human subject an amount of rapamycin effective totreat, prevent, inhibit, delay onset of, or cause regression of wet AMD.

As described in further detail in the Detailed Description section, themethods, compositions and liquid formulations may also be used fordelivery to a subject, including but not limited to a human subject orto the eye of a human subject of therapeutically effective amounts ofrapamycin for the treatment, prevention, inhibition, delaying of theonset of, or causing the regression of wet AMD. In some variations, themethods, compositions, and liquid formulations are used to treat wetAMD. In some variations, the methods, compositions, and liquidformulations are used to prevent wet AMD. In some variations, themethods and formulations described herein are used to prevent thetransition from dry AMD to wet AMD. The methods, compositions and liquidformulations may also be used for delivery to a subject, including butnot limited to a human subject or to the eye of a subject oftherapeutically effective amounts of rapamycin for the treatment,prevention, inhibition, delaying of the onset of, or causing theregression of CNV. In some variations, the methods, compositions andliquid formulations are used to treat CNV. The methods, compositions andliquid formulations may also be used for delivery to a subject,including but not limited to a human subject or to the eye of a subjectof therapeutically effective amounts of rapamycin for the treatment,prevention, inhibition, delaying of the onset of, or causing theregression of angiogenesis in the eye. In some variations, the methods,compositions and liquid formulations are used to treat angiogenesis.Other diseases and conditions that may be treated, prevented, inhibited,have onset delayed, or caused to regress using rapamycin are describedin the Diseases and Conditions section of the Detailed Description.

As described in further detail in the Detailed Description, the methods,compositions and liquid formulations may also be used for delivery to asubject, including but not limited to a human subject or to the eye of asubject of therapeutically effective amounts of therapeutic agents otherthan rapamycin for the treatment, prevention, inhibition, delaying ofthe onset of, or causing the regression of wet AMD. In some variations,the methods, compositions and liquid formulations are used to treat wetAMD. Therapeutic agents that may be used are described in detail in theTherapeutic Agents section. Such therapeutic agents include but are notlimited to immunophilin binding compounds. Immunophilin bindingcompounds that may be used include but are not limited to the limusfamily of compounds described further in the Therapeutic Agents sectionherein, including rapamycin, SDZ-RAD, tacrolimus, everolimus,pimecrolimus, CCI-779, AP23841, ABT-578, derivatives, analogs, prodrugs,salts and esters thereof. The methods, compositions and liquidformulations may also be used for delivery to a subject, including butnot limited to a human subject or to the eye of a subject oftherapeutically effective amounts of therapeutic agents for thetreatment, prevention, inhibition, delaying of the onset of, or causingthe regression of CNV. In some variations, the methods, compositions andliquid formulations are used to treat CNV. The methods, compositions andliquid formulations may also be used for delivery to a subject,including but not limited to a human subject or to the eye of a subjectof therapeutically effective amounts of therapeutic agents for thetreatment, prevention, inhibition, delaying of the onset of, or causingthe regression of angiogenesis in the eye. In some variations, themethods, compositions and liquid formulations are used to treatangiogenesis. Other diseases and conditions that may be treated,prevented, inhibited, have onset delayed, or caused to regress usingtherapeutic agents other than rapamycin are described in the Diseasesand Conditions section of the Detailed Description.

One liquid formulation described herein comprises a solution thatincludes a therapeutic agent dissolved in a solvent. Generally, anysolvent that has the desired effect may be used in which the therapeuticagent dissolves and which can be administered to a subject, includingbut not limited to a human subject or an eye of a subject. Generally,any concentration of therapeutic agent that has the desired effect canbe used. The formulation in some variations is a solution which isunsaturated, a saturated or a supersaturated solution. The solvent maybe a pure solvent or may be a mixture of liquid solvent components. Insome variations the solution formed is an in situ gelling formulation.Solvents and types of solutions that may be used are well known to thoseversed in such drug delivery technologies.

The liquid formulations described herein may form a non-dispersed masswhen placed into a rabbit eye, including but not limited to the vitreousof a rabbit eye. In some variations the non-dispersed mass comprises agel. In some variations, the liquid formulation comprises a therapeuticagent and a plurality of polymers. In some variations one of thepolymers is polyacrylate or polymethacrylate. In some variations one ofthe polymers is polyvinylpyrrolidone.

In some variations, the non-dispersed mass comprises a depot. In somevariations, the non-dispersed mass consists of a depot.

For liquid formulations which form a non-dispersed mass, thenon-dispersed mass may generally be any geometry or shape. Thenon-dispersed mass-forming liquid formulations may, for instance, appearas a compact spherical mass when placed in the vitreous. In somevariations the liquid formulations described herein form a milky orwhitish colored semi-contiguous or semi-solid non-dispersed massrelative to the medium in which it is placed, when placed in thevitreous.

The liquid formulations may generally be administered in any volume thathas the desired effect. In one method a volume of a liquid formulationis administered to the vitreous and the liquid formulation is less thanone half the volume of the vitreous.

Routes of administration that may be used to administer a liquidformulation include but are not limited to (1) placement of the liquidformulation by placement, including by injection, into a medium,including but not limited to an aqueous medium in the body, includingbut not limited to intraocular or periocular injection; or (2) oraladministration of the liquid formulation. The liquid formulation may beadministered systemically, including but not limited to the followingdelivery routes: rectal, vaginal, infusion, intramuscular,intraperitoneal, intraarterial, intrathecal, intrabronchial,intracisternal, cutaneous, subcutaneous, intradermal, transdermal,intravenous, intracervical, intraabdominal, intracranial,intrapulmonary, intrathoracic, intratracheal, nasal, buccal, sublingual,oral, parenteral, or nebulised or aerosolized using aerosol propellants.In some variations, the liquid formulation is administeredsubconjunctivally. In some variations, the liquid formulation isadministered intravitreally.

The liquid formulations described herein may be delivered to any mediumof a subject, including but not limited to a human subject, includingbut not limited to an aqueous medium of a subject.

One liquid formulation described herein comprises a liquid formulationof rapamycin or other therapeutic agent. The liquid formulations maycomprise a solution, suspension, an in situ gelling formulation, or anemulsion. The droplets in the emulsion may generally be of any size,including but not limited to up to about 5,000 nm.

In some formulations described herein, the liquid formulations maycomprise a therapeutic agent including but not limited to rapamycin, andone or more solubilizing agents or solvents. In some variations, thesolubilizing agent or solvent is glycerin, DMSO, DMA,N-methylpyrrolidone, ethanol, benzyl alcohol, isopropyl alcohol,polyethylene glycol of various molecular weights, including but notlimited to PEG 300 and PEG 400, or propylene glycol or a mixture of oneor more thereof.

In some formulations described herein, the liquid formulation includeshyaluronic acid.

The liquid formulations described herein may deliver a therapeutic agentor agents for an extended period of time. One nonlimiting example ofsuch an extended release delivery system is a liquid formulation thatdelivers a therapeutic agent or agents to a subject, including but notlimited to a human subject or to the eye of a subject in an amountsufficient to maintain an amount effective to treat, prevent, inhibit,delay onset of, or cause regression of a disease or condition in asubject for an extended period of time. In some variations, the liquidformulation is used to treat a disease or condition in a subject,including but not limited to a human subject. In some variations, theliquid formulation delivers the therapeutic agent for at least aboutone, about two, about three, about six, about nine, or about twelvemonths.

The liquid formulations described herein may deliver rapamycin or othertherapeutic agents for an extended period of time. One nonlimitingexample of such an extended release delivery system is a liquidformulation that delivers rapamycin to a subject, including but notlimited to a human subject or to the eye of a subject in an amountsufficient to maintain an amount effective to treat, prevent, inhibit,delay onset of, or cause regression of wet age-related maculardegeneration for an extended period of time. In some variations, theliquid formulation is used to treat wet age-related macular degenerationfor an extended period of time. In some variations, the liquidformulation is used to prevent wet age-related macular degeneration foran extended period of time. In some variations, the liquid formulationis used to prevent transition of dry AMD to wet AMD for an extendedperiod of time. In one nonlimiting example, the liquid formulationdelivers the rapamycin to the vitreous, sclera, retina, choroid, macula,or other tissues of a subject, including but not limited to a humansubject in an amount sufficient to treat, prevent, inhibit, delay onsetof, or cause regression of wet age-related macular degeneration for atleast about three, about six, about nine, or about twelve months. Insome variations, the level of rapamycin is sufficient to treat AMD. Insome variations, the level of rapamycin is sufficient to prevent onsetof wet AMD.

Other extended periods of release are described in the DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C schematically depicts formation of a non-dispersed mass,after injection of a liquid formulation into the vitreous of an eye, asit is believed to occur in some variations.

FIG. 2 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid (ng/mg), and sclera (ng/mg) of rabbit eyes at 20, 40, 67, and 90days after subconjunctival injection of a 1.256% solution of rapamycinin water, ethanol, and F127 (Lutrol).

FIG. 3 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid (ng/mg), and sclera (ng/mg) of rabbit eyes at 14, 35, 62, and 85days after subconjunctival injection of a 5% solution of rapamycin inPEG 400 and ethanol. The level of rapamycin present in the vitreous(ng/ml) is also shown at 2 days after injection.

FIG. 4 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid (ng/mg), and sclera (ng/mg) of rabbit eyes at 14, 35, 62, and 90days after intravitreal injection of a 5% solution of rapamycin in PEG400 and ethanol. The level of rapamycin present in the vitreous (ng/ml)is also shown at 2 days after injection.

FIG. 5A-5C depict images of rabbit eyes 8 days after intravitrealinjection of 10 μl (FIG. 5A), 20 μl (FIG. 5B), and 40 μl (FIG. 5C) of a6% rapamycin suspension in PEG400.

FIG. 6 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid tissues (ng/mg), and sclera (ng/mg) of rabbit eyes at 7, 32, 45,and 90 days after subconjunctival injection of a 4.2% solution ofrapamycin in ethanol, PVP K90, PEG 400, and Eudragit RL 100.

FIG. 7 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid tissues (ng/mg), and sclera (ng/mg) of rabbit eyes at 14, 42,63, and 91 days after subconjunctival injection of a 3% suspension ofrapamycin in PEG 400.

FIG. 8 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid tissues (ng/mg) and sclera (ng/mg) of rabbit eyes at 14, 42, 63,and 91 days after intravitreal injection of a 3% suspension of rapamycinin PEG 400, and in the vitreous at 63 and 91 days after injection.

FIG. 9 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid tissues (ng/mg), and sclera (ng/mg) of rabbit eyes at 14, 42,63, and 91 days after subconjunctival injection of a 2% solution ofrapamycin in ethanol and PEG 400.

FIG. 10 depicts the level of rapamycin in the retina choroid tissues(ng/mg) and sclera (ng/mg) of rabbit eyes at 14, 42, 63, and 91 daysafter intravitreal injection of a 2% solution of rapamycin in ethanoland PEG 400.

FIG. 11 depicts the level of rapamycin in the vitreous (ng/ml) of rabbiteyes at 63 and 91 days after intravitreal injection of a 2% solution ofrapamycin in ethanol and PEG 400.

FIG. 12 depicts the level of rapamycin in the vitreous (ng/ml) of rabbiteyes at 5, 30, 60, 90, and 120 days after subconjunctival injection of20 μl, 40 μl, and 60 μl doses of a 2% solution of rapamycin in ethanoland PEG 400.

FIG. 13 depicts the level of rapamycin in the retina choroid tissues(ng/mg) of rabbit eyes at 5, 30, 60, 90, and 120 days aftersubconjunctival injection of 20 μl, 40 μl, and 60 μl doses of a 2%solution of rapamycin in ethanol and PEG 400.

FIG. 14 depicts the level of rapamycin in the vitreous (ng/ml) of rabbiteyes at 5, 30, 60, 90, and 120 days after intravitreal injection of 20μl and 40 μl doses of a 2% solution of rapamycin in ethanol and PEG 400and of a 100 μl dose of a 0.4% rapamycin solution in ethanol and PEG400.

FIG. 15 depicts the level of rapamycin in the retina choroid tissues(ng/mg) of rabbit eyes at 5, 30, 60, 90, and 120 days after intravitrealinjection of 20 μl and 40 μl doses of a 2% solution of rapamycin inethanol and PEG 400 and of a 100 μl dose of a 0.4% rapamycin solution inethanol and PEG 400.

FIG. 16 depicts the level of rapamycin in the vitreous (ng/ml) of rabbiteyes at 5 and 14 days after subconjunctival injection of a single 10 μldose, a single 60 μl dose, two 30 μl doses, and three 30 μl doses of a2% solution of rapamycin in ethanol and PEG 400.

FIG. 17 depicts the level of rapamycin in the retina choroid tissues(ng/mg) of rabbit eyes at 5 and 14 days after subconjunctival injectionof a single 10 μl dose, a single 60 μl dose, two 30 μl doses, and three30 μl doses of a 2% solution of rapamycin in ethanol and PEG 400.

FIG. 18 depicts the level of rapamycin in the vitreous (ng/ml) of rabbiteyes at 5, 14, and 30 days after subconjunctival injection of a single10 μl dose, a single 30 μl dose, and three 30 μl doses of a 3%suspension of rapamycin in PEG 400.

FIG. 19 depicts the level of rapamycin in the retina choroid tissues(ng/mg) of rabbit eyes at 5, 14, and 30 days after subconjunctivalinjection of a single 10 μl dose, a single 30 μl dose, and three 30 μldoses of a 3% suspension of rapamycin in PEG 400.

FIG. 20 depicts the level of rapamycin in the retina choroid tissues(ng/mg) of rabbit eyes at 5, 30, and 90 days after intravitrealinjection of 10 μl of a 0.2% solution of rapamycin in ethanol and PEG400, of 10 μl of a 0.6% solution of rapamycin in ethanol and PEG 400,and of 10 μl of a 2% solution of rapamycin in ethanol and PEG 400.

FIG. 21 depicts the level of rapamycin in the vitreous (ng/ml) of rabbiteyes at 5, 30, and 90 days after intravitreal injection of 10 μl of a0.2% solution of rapamycin in ethanol and PEG 400, of 10 μl of a 0.6%solution of rapamycin in ethanol and PEG 400, and of 10 μl of a 2%solution of rapamycin in ethanol and PEG 400.

FIG. 22 depicts the level of rapamycin in the aqueous humor (ng/ml) ofrabbit eyes, the cornea (ng/mg), and the retina choroid tissues (ng/mg)at 1, 4, 7, 11, 14, 21, 28, 35, 54, and 56 days after subconjunctivalinjection of 40 μl of a 2% solution of rapamycin in ethanol and PEG 400.

DETAILED DESCRIPTION

Described herein are compositions, liquid formulations and methodsrelating to delivery of therapeutic agents to a subject, including butnot limited to a human subject or to the eye of a subject. Thesecompositions, liquid formulations, and methods may be used for thetreatment, prevention, inhibition, delaying onset of, or causingregression of diseases and conditions of the eye including but notlimited to diseases or conditions of the posterior segment, includingbut not limited to choroidal neovascularization; macular degeneration;age-related macular degeneration, including wet AMD and dry AMD; retinalangiogenesis; chronic uveitis; and other retinoproliferative conditions.In some variations, the compositions, liquid formulations, and methodsare used for the treatment of the aforementioned diseases or conditionsof the eye.

Herein are described (1) the therapeutic agents that may be delivered toa subject, including but not limited to a human subject or an eye of asubject using the compositions, liquid formulations, and methodsdescribed herein, (2) the diseases and conditions that may be treated,prevented, inhibited, onset delayed, or regression caused by delivery ofthe therapeutic agents, (3) liquid formulations that may be used todeliver the therapeutic agents, (4) routes of administration fordelivery of the liquid formulations, (5) extended delivery oftherapeutic agents including but not limited to rapamycin, and (6)description of the treatment of CNV and wet AMD by delivery of rapamycinto a subject, including but not limited to a human subject or to the eyeof a subject for an extended period of time using the describedcompositions and liquid formulations.

The term “about,” as used herein, generally refers to the level ofaccuracy that is obtained when the methods described herein, such as themethods in the examples, are used. However, by “about” a certain amountof a component of a formulation is meant 90-110% of the amount stated.

Therapeutic Agents

Most generally, any compounds and compositions currently known or yet tobe discovered that are useful in treating, preventing, inhibiting,delaying the onset of, or causing the regression of the diseases andconditions described herein may be therapeutic agents for use in thecompositions, liquid formulations, and methods described herein.

Therapeutic agents that may be used include compounds that act bybinding members of the immunophilin family of cellular proteins. Suchcompounds are known as “immunophilin binding compounds.” Immunophilinbinding compounds include but are not limited to the “limus” family ofcompounds. Examples of limus compounds that may be used include but arenot limited to cyclophilins and FK506-binding proteins (FKBPs),including sirolimus (rapamycin) and its water soluble analog SDZ-RAD(Novartis), TAFA-93 (Isotechnika), tacrolimus, everolimus, RAD-001(Novartis), pimecrolimus, temsirolimus, CCI-779 (Wyeth), AP23841(Ariad), AP23573 (Ariad), and ABT-578 (Abbott Laboratories). Limuscompound analogs and derivatives that may be used include but are notlimited to the compounds described in U.S. Pat. Nos. 5,527,907;6,376,517; and 6,329,386 and U.S. patent application Ser. No.09/950,307, each of which is incorporated herein by reference in theirentirety. Therapeutic agents also include analogs, prodrugs, salts andesters of limus compounds.

The terms rapamycin, rapa, and sirolimus are used interchangeablyherein.

Other rapamycin derivatives that may be used include, withoutlimitation, 7-epi-rapamycin, 7-thiomethyl-rapamycin,7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin,7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-desmethyl-rapamycin,mono- and di-ester derivatives of rapamycin, 27-oximes of rapamycin;42-oxo analog of rapamycin; bicyclic rapamycins; rapamycin dimers; silylethers of rapamycin; rapamycin arylsulfonates and sulfamates,mono-esters and di-esters at positions 31 and 42, 30-demethoxyrapamycin, and other derivatives described in Vezina et al., “Rapamycin(AY-22,989), A New Antifungal Antibiotic. I. Taxonomy Of The ProducingStreptomycete And Isolation Of The Active Principle” J. Antibiot.(Tokyo) 28:721-726 (1975); Sehgal et al., “Rapamycin (AY-22,989), A NewAntifungal Antibiotic. II. Fermentation, Isolation And Characterization”J. Antibiot. (Tokyo) 28:727-732 (1975); Sehgal et al.,“Demethoxyrapamycin (AY-24,668), A New Antifungal Antibiotic” J.Antibiot. (Tokyo) 36:351-354 (1983); and Paiva et al., “Incorporation OfAcetate, Propionate, And Methionine Into Rapamycin By Streptomyceteshygroscopicus” J Nat Prod 54:167-177 (1991), WO 92/05179, EP 467606,Caufield et al., “Hydrogenated Rapamycin Derivatives” U.S. Pat. No.5,023,262; Kao et al., “Bicyclic Rapamycins” U.S. Pat. No. 5,120,725;Kao et al., “Rapamycin Dimers” U.S. Pat. No. 5,120,727; Failli et al.,“Silyl Ethers Of Rapamycin” U.S. Pat. No. 5,120,842; Failli et al.,“Rapamycin 42-Sulfonates And 42-(N-carboalkoxy) Sulfamates Useful AsImmunosuppressive Agents” U.S. Pat. No. 5,177,203; Nicolaou et al.,“Total Synthesis Of Rapamycin” J. Am. Chem. Soc. 115: 4419-4420 (1993);Romo et al, “Total Synthesis Of (−) Rapamycin Using An Evans-TishchenkoFragment Coupling” J. Am. Chem. Soc. 115:7906-7907 (1993); and Haywardet al, “Total Synthesis Of Rapamycin Via A Novel Titanium-Mediated AldolMacrocyclization Reaction” J. Am. Chem. Soc., 115:9345-9346 (1993), eachof which is incorporated herein by reference in its entirety.

The limus family of compounds may be used in the compositions, liquidformulations and methods for the treatment, prevention, inhibition,delaying the onset of, or causing the regression ofangiogenesis-mediated diseases and conditions of the eye, includingchoroidal neovascularization. The limus family of compounds may be usedto prevent, treat, inhibit, delay the onset of, or cause regression ofAMD, including wet AMD. Rapamycin and rapamycin derivatives and analogsmay be used to prevent, treat, inhibit, delay the onset of, or causeregression of angiogenesis-mediated diseases and conditions of the eye,including choroidal neovascularization. Rapamycin may be used toprevent, treat, inhibit, delay the onset of, or cause regression of AMD,including wet AMD. In some variations, a member of the limus family ofcompounds or rapamycin is used to treat wet AMD or angiogenesis-mediateddiseases and conditions of the eye including choroidalneovascularization.

Other therapeutic agents that may be used include those disclosed in thefollowing patents and publications, the contents of each of which isincorporated herein by reference in its entirety: PCT publication WO2004/027027, titled Method of inhibiting choroidal neovascularization,assigned to Trustees of the University of Pennsylvania; U.S. Pat. No.5,387,589, titled Method of Treating Ocular Inflammation, assigned toUniversity of Louisville Research Foundation; U.S. Pat. No. 6,376,517,titled Pipecolic acid derivatives for vision and memory disorders,assigned to GPI NIL Holdings, Inc; PCT publication WO 2004/028477,titled Method subretinal administration of therapeutics includingsteroids: method for localizing pharmadynamic action at the choroid andretina; and related methods for treatment and or prevention of retinaldiseases, assigned to Innorx, Inc; U.S. Pat. No. 6,416,777, titledOphthalmic drug delivery device, assigned to Alcon Universal Ltd; U.S.Pat. No. 6,713,081, titled Ocular therapeutic agent delivery device andmethods for making and using such devices, assigned to Department ofHealth and Human Services; U.S. Pat. No. 5,100,899, titled Methods ofinhibiting transplant rejection in mammals using rapamycin andderivatives and prodrugs thereof.

Other therapeutic agents that may be used include pyrrolidine,dithiocarbamate (NFκB inhibitor); squalamine; TPN 470 analogue andfumagillin; PKC (protein kinase C) inhibitors; Tie-1 and Tie-2 kinaseinhibitors; inhibitors of VEGF receptor kinase; proteosome inhibitorssuch as Velcade™ (bortezomib, for injection; ranibuzumab (Lucentis™) andother antibodies directed to the same target; pegaptanib (Macugen™);vitronectin receptor antagonists, such as cyclic peptide antagonists ofvitronectin receptor-type integrins; α-v/β-3 integrin antagonists;α-v/β-1 integrin antagonists; thiazolidinediones such as rosiglitazoneor troglitazone; interferon, including γ-interferon or interferontargeted to CNV by use of dextran and metal coordination; pigmentepithelium derived factor (PEDF); endostatin; angiostatin; tumistatin;canstatin; anecortave acetate; acetonide; triamcinolone;tetrathiomolybdate; RNA silencing or RNA interference (RNAi) ofangiogenic factors, including ribozymes that target VEGF expression;Accutane™ (13-cis retinoic acid); ACE inhibitors, including but notlimited to quinopril, captopril, and perindozril; inhibitors of mTOR(mammalian target of rapamycin); 3-aminothalidomide; pentoxifylline;2-methoxyestradiol; colchicines; AMG-1470; cyclooxygenase inhibitorssuch as nepafenac, rofecoxib, diclofenac, rofecoxib, NS398, celecoxib,vioxx, and (E)-2-alkyl-2(4-methanesulfonylphenyl)-1-phenylethene; t-RNAsynthase modulator; metalloprotease 13 inhibitor; acetylcholinesteraseinhibitor; potassium channel blockers; endorepellin; purine analog of6-thioguanine; cyclic peroxide ANO-2; (recombinant) arginine deiminase;epigallocatechin-3-gallate; cerivastatin; analogues of suramin; VEGFtrap molecules; apoptosis inhibiting agents; Visudyne™, snET2 and otherphoto sensitizers, which may be used with photodynamic therapy (PDT);inhibitors of hepatocyte growth factor (antibodies to the growth factoror its receptors, small molecular inhibitors of the c-met tyrosinekinase, truncated versions of HGF e.g. NK4).

Other therapeutic agents that may be used include anti-inflammatoryagents, including, but not limited to nonsteroidal anti-inflammatoryagents and steroidal anti-inflammatory agents. In some variations,active agents that may be used in the liquid formulations areace-inhibitors, endogenous cytokines, agents that influence basementmembrane, agents that influence the growth of endothelial cells,adrenergic agonists or blockers, cholinergic agonists or blockers,aldose reductase inhibitors, analgesics, anesthetics, antiallergics,antibacterials, antihypertensives, pressors, antiprotozoal agents,antiviral agents, antifungal agents, anti-infective agents, antitumoragents, antimetabolites, and antiangiogenic agents.

Steroidal therapeutic agents that may be used include but are notlimited to 21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol,clobetasone, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide, and any of their derivatives.

In some variations, cortisone, dexamethasone, fluocinolone,hydrocortisone, methylprednisolone, prednisolone, prednisone, andtriamcinolone, or their derivatives, may be used. The liquid formulationmay include a combination of two or more steroidal therapeutic agents.

In one nonlimiting example, the steroidal therapeutic agents mayconstitute from about 0.05% to about 50% by weight of the liquidformulation. In another nonlimiting example, the steroid constitutesfrom about 0.05% to about 10%, between about 10% to about 20%; betweenabout 30% to about 40%; or between about 40% to about 50% by weight ofthe liquid formulation.

Other nonlimiting examples of therapeutic agents that may be usedinclude but are not limited to anaesthetics, analgesics, celltransport/mobility impending agents such as colchicines, vincristine,cytochalasin B and related compounds; carbonic anhydrase inhibitors suchas acetazolamide, methazolamide, dichlorphenamide, diamox andneuroprotectants such as nimodipine and related compounds; antibioticssuch as tetracycline, chlortetracycline, bacitracin, neomycin,polymyxin, gramicidin, cephalexin, oxytetracycline, chloramphenicol,rifampicin, ciprofloxacin, aminosides, gentamycin, erythromycin andpenicillin, quinolone, ceftazidime, vancomycine imipeneme; antifungalssuch as amphotericin B, fluconazole, ketoconazole and miconazole;antibacterials such as sulfonamides, sulfadiazine, sulfacetamide,sulfamethizole and sulfisoxazole, nitrofurazone and sodium propionate;antivirals, such as idoxuridine, trifluorothymidine, trifluorouridine,acyclovir, ganciclovir, cidofovir, interferon, DDI, AZT, foscamet,vidarabine, irbavirin, protease inhibitors and anti-cytomegalovirusagents; antiallergenics such as sodium cromoglycate, antazoline,methapyriline, chlorpheniramine, cetirizine, pyrilamine andprophenpyridamine; synthetic gluocorticoids and mineralocorticoids andmore generally hormones forms derivating from the cholesterol metabolism(DHEA, progesterone, estrogens); non-steroidal anti-inflammatories suchas salicylate, indomethacin, ibuprofen, diclofenac, flurbiprofen,piroxicam and COX2 inhibitors; antineoplastics such as carmustine,cisplatin, fluorouracil; adriamycin, asparaginase, azacitidine,azathioprine, bleomycin, busulfan, carboplatin, carmustine,chlorambucil, cyclophosphamide, cyclosporine, cytarabine, dacarbazine,dactinomycin, daunorubicin, doxorubicin, estramustine, etoposide,etretinate, filgrastin, floxuridine, fludarabine, fluorouracil,florxymesterone, flutamide, goserelin, hydroxyurea, ifosfamide,leuprolide, levamisole, limustine, nitrogen mustard, melphalan,mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin,pipobroman, plicamycin, procarbazine, sargramostin, streptozocin,tamoxifen, taxol, teniposide, thioguanine, uracil mustard, vinblastine,vincristine and vindesine; immunological drugs such as vaccines andimmune stimulants; insulin, calcitonin, parathyroid hormone and peptideand vasopressin hypothalamus releasing factor; beta adrenergic blockerssuch as timolol, levobunolol and betaxolol; cytokines, interleukines andgrowth factors epidermal growth factor, fibroblast growth factor,platelet derived growth factor, transforming growth factor beta, ciliaryneurotrophic growth factor, glial derived neurotrophic factor, NGF, EPO,PLGF, brain nerve growth factor (BNGF), vascular endothelial growthfactor (VEGF) and monoclonal antibodies or fragments thereof directedagainst such growth factors; anti-inflammatories such as hydrocortisone,dexamethasone, fluocinolone, prednisone, prednisolone,methylprednisolone, fluorometholone, betamethasone and triamcinolone;decongestants such as phenylephrine, naphazoline and tetrahydrazoline;miotics and anti-cholinesterases such as pilocarpine, carbachol,di-isopropyl fluorophosphate, phospholine iodine and demecarium bromide;mydriatics such as atropine sulphate, cyclopentolate, homatropine,scopolamine, tropicamide, eucatropine; sympathomimetics such asepinephrine and vasoconstrictors and vasodilators, anticlotting agentssuch as heparin, antifibrinogen, fibrinolysin, anticlotting activase,antidiabetic agents include acetohexamide, chlorpropamide, glipizide,glyburide, tolazamide, tolbutamide, insulin and aldose reductaseinhibitors, hormones, peptides, nucleic acids, saccharides, lipids,glycolipids, glycoproteins and other macromolecules include endocrinehormones such as pituitary, insulin, insulin-related growth factor,thyroid, growth hormones; heat shock proteins; immunological responsemodifiers such as muramyl dipeptide, cyclosporins, interferons(including alpha-, beta- and gamma-interferons), interleukin-2,cytokines, FK506 (an epoxy-pyrido-oxaazcyclotricosine-tetrone, alsoknown as Tacrolimus), tumor necrosis factor, pentostatin, thymopentin,transforming factor beta2, erythropoetin; antineogenesis proteins (e.g.anti VEGF, interferons), antibodies (monoclonal, polyclonal, humanized,etc.) or antibodies fragments, oligoaptamers, aptamers and genefragments (oligonucleotides, plasmids, ribozymes, small interference RNA(SiRNA), nucleic acid fragments, peptides), immunomodulators such asendoxan, thalidomide, tamoxifene; antithrombolytic and vasodilatoragents such as rtPA, urokinase, plasmin; nitric oxide donors, nucleicacids, dexamethasone, cyclosporin A, azathioprine, brequinar,gusperimus, 6-mercaptopurine, mizoribine, rapamycin, tacrolimus(FK-506), folic acid analogs (e.g., denopterin, edatrexate,methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate), purineanalogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine,thiaguanine), pyrimidine analogs (e.g., ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, doxifluridine, emitefur,enocitabine, floxuridine, fluorouracil, gemcitabine, tegafur)fluocinolone, triaminolone, anecortave acetate, fluorometholone,medrysone, and prednislone. In some variations the immunosuppressiveagent is dexamethasone. In some variations the immunosuppressive agentis cyclosporin A.

In some variations the formulation comprises a combination of one ormore therapeutic agents.

Other nonlimiting examples of therapeutic agents that may be used in theformulations described herein include antibacterial antibiotics,aminoglycosides (e.g., amikacin, apramycin, arbekacin, bambermycins,butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), gentamicin,isepamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate,netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin,streptomycin, tobramycin, trospectomycin), amphenicols (e.g.,azidamfenicol, chloramphenicol, florfenicol, thiamphenicol), ansamycins(e.g., rifamide, rifampin, rifamycin sv, rifapentine, rifaximin),P-lactams (e.g., carbacephems (e.g., loracarbef), carbapenems (e.g.,biapenem, imipenem, meropenem, panipenem), cephalosporins (e.g.,cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin,cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime, cefetamet,cefixime, cefinenoxime, cefodizime, cefonicid, cefoperazone, ceforanide,cefotaxime, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome,cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime,cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime,cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin,cephaloridine, cephalosporin, cephalothin, cephapirin sodium,cephradine, pivcefalexin), cephamycins (e.g., cefbuperazone,cefinetazole, cefminox, cefotetan, cefoxitin), monobactams (e.g.,aztreonam, carumonam, tigemonam), oxacephems, flomoxef, moxalactam),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,carbenicillin, carindacillin, clometocillin, cloxacillin, cyclacillin,dicloxacillin, epicillin, fenbenicillin, floxacillin, hetacillin,lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillinsodium, oxacillin, penamecillin, penethamate hydriodide, penicillin gbenethamine, penicillin g benzathine, penicillin g benzhydrylamine,penicillin g calcium, penicillin g hydrabamine, penicillin g potassium,penicillin g procaine, penicillin n, penicillin o, penicillin v,penicillin v benzathine, penicillin v hydrabamine, penimepicycline,phenethicillin potassium, piperacillin, pivampicillin, propicillin,quinacillin, sulbenicillin, sultamicillin, talampicillin, temocillin,ticarcillin), ritipenem, lincosamides (e.g., clindamycin, lincomycin),macrolides (e.g., azithromycin, carbomycin, clarithromycin,dirithromycin, erythromycin, erythromycin acistrate, erythromycinestolate, erythromycin glucoheptonate, erythromycin lactobionate,erythromycin propionate, erythromycin stearate, josamycin, leucomycins,midecamycins, miokamycin, oleandomycin, primycin, rokitamycin,rosaramicin, roxithromycin, spiramycin, troleandomycin), polypeptides(e.g., amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, fusafungine, gramicidin s, gramicidin(s), mikamycin,polymyxin, pristinamycin, ristocetin, teicoplanin, thiostrepton,tuberactinomycin, tyrocidine, tyrothricin, vancomycin, viomycin,virginiamycin, zinc bacitracin), tetracyclines (e.g., apicycline,chlortetracycline, clomocycline, demeclocycline, doxycycline,guamecycline, lymecycline, meclocycline, methacycline, minocycline,oxytetracycline, penimepicycline, pipacycline, rolitetracycline,sancycline, tetracycline), and others (e.g., cycloserine, mupirocin,tuberin); synthetic antibacterials, 2.4-Diaminopyrimidines (e.g.,brodimoprim, tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone,furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol,nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs(e.g., cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin,fleroxacin, flumequine, grepafloxacin, lomefloxacin, miloxacin,nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid,pazufloxacin, pefloxacin, pipemidic acid, piromidic acid, rosoxacin,rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin),sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide,chloramine-b, chloramine-t, dichloramine t, n2-formylsulfisomidine,n4-β-d-glucosylsulfanilamide, mafenide, 4′-(methylsulfamoyl)sulfanilanilide, noprylsulfamide, phthalylsulfacetamide,phthalylsulfathiazole, salazosulfadimidine, succinylsulfathiazole,sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine,sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine,sulfadoxine, sulfaethidole, sulfaguanidine, sulfaguanol, sulfalene,sulfaloxic acid, sulfamerazine, sulfameter, sulfamethazine,sulfamethizole, sulfamethomidine, sulfamethoxazole,sulfamethoxypyridazine, sulfametrole, sulfamidochrysoidine, sulfamoxole,sulfanilamide, 4-sulfanilamidosalicylic acid,n4-sulfanilylsulfanilamide, sulfanilylurea, n-sulfanilyl-3,4-xylamide,sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine,sulfapyridine, sulfasomizole, sulfasymazine, sulfathiazole,sulfathiourea, sulfatolamide, sulfisomidine, sulfisoxazole) sulfones(e.g., acedapsone, acediasulfone, acetosulfone sodium, dapsone,diathymosulfone, glucosulfone sodium, solasulfone, succisulfone,sulfanilic acid, p-sulfanilylbenzylamine, sulfoxone sodium,thiazolsulfone), and others (e.g., clofoctol, hexedine, methenamine,methenamine anhydromethylene-citrate, methenamine hippurate, methenaminemandelate, methenamine sulfosalicylate, nitroxoline, taurolidine,xibomol), antifungal antibiotics, polyenes (e.g., amphotericin b,candicidin, dermostatin, filipin, fungichromin, hachimycin, hamycin,lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin),azaserine, griseofulvin, oligomycins, neomycin undecylenate,pyrrolnitrin, siccanin, tubercidin, viridin, synthetic antifungals,allylamines (e.g., butenafine, naftifine, terbinafine), imidazoles(e.g., bifonazole, butoconazole, chlordantoin, chlormidazole,cloconazole, clotrimazole, econazole, enilconazole, fenticonazole,flutrimazole, isoconazole, ketoconazole, lanoconazole, miconazole,omoconazole, oxiconazole nitrate, sertaconazole, sulconazole,tioconazole), thiocarbamates (e.g., tolciclate, tolindate, tolnaftate),triazoles (e.g., fluconazole, itraconazole, saperconazole, terconazole),acrisorcin, amorolfine, biphenamine, bromosalicylchloranilide,buclosamide, calcium propionate, chlorphenesin, ciclopirox, cloxyquin,coparaffinate, diamthazole dihydrochloride, exalarnide, flucytosine,halethazole, hexetidine, loflucarban, nifuratel, potassium iodide,propionic acid, pyrithione, salicylanilide, sodium propionate,sulbentine, tenonitrozole, triacetin, ujothion, undecylenic acid, zincpropionate, antineoplastics, antibiotics and analogs (e.g.,aclacinomycins, actinomycin f1, anthramycin, azaserine, bleomycins,cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin,daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycin,olivomycines, peplomycin, pirarubicin, plicamycin, porfiromycin,puromycin, streptonigrin, streptozocin, tubercidin, zinostatin,zorubicin), antimetabolites (e.g. folic acid analogs (e.g., denopterin,edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®,trimetrexate), purine analogs (e.g., cladribine, fludarabine,6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogs (e.g.,ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil,gemcitabine, tagafur), antiinflammatory agents, steroidalantiinflammatory agents, acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clobetasone, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacort, desonide, desoximetasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin butyl, fluocortolone,fluorometholone, fluperolone acetate, fluprednidene acetate,fluprednisolone, flurandrenolide, fluticasone propionate, formocortal,halcinonide, halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, and triamcinolonehexacetonide, non-steroidal antiinflammatory agents, aminoarylcarboxylicacid derivatives (e.g., enfenamic acid, etofenamate, flufenamic acid,isonixin, meclofenamic acid, mefenamic acid, niflumic acid,talniflumate, terofenamate, tolfenamic acid), arylacetic acidderivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac,amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac,diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin,sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acidderivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin),arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine),arylpropionic acid derivatives (e.g., alminoprofen, benoxaprofen,bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen,flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen,naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinicacid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles(e.g., difenamizole, epirizole), pyrazolones (e.g., apazone,benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone,phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone,thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol,aspirin, benorylate, bromosaligenin, calcium acetylsalicylate,diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate,imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholinesalicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenylacetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-aceticacid, salicylsulfuric acid, salsalate, sulfasalazine),thiazinecarboxamides (e.g., ampiroxicam, droxicam, isoxicam, lomoxicam,piroxicam, tenoxicam), ε-acetamidocaproic acid, s-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,a-bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol,guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline, perisoxal,proquazone, superoxide dismutase, tenidap, and zileuton.

The therapeutic agents may also be used in combination with othertherapeutic agents and therapies, including but not limited to agentsand therapies useful for the treatment, prevention, inhibition, delayingonset of, or causing regression of angiogenesis or neovascularization,particularly CNV. In some variations the additional agent or therapy isused to treat regression of angiogenesis or neovascularization,particularly CNV. Non-limiting examples of such additional agents andtherapies include pyrrolidine, dithiocarbamate (NFκB inhibitor);squalamine; TPN 470 analogue and fumagillin; PKC (protein kinase C)inhibitors; Tie-1 and Tie-2 kinase inhibitors; inhibitors of VEGFreceptor kinase; proteosome inhibitors such as Velcade™ (bortezomib, forinjection; ranibuzumab (Lucentis™) and other antibodies directed to thesame target; pegaptanib (Macugen™); vitronectin receptor antagonists,such as cyclic peptide antagonists of vitronectin receptor-typeintegrins; α-v/β-3 integrin antagonists; α-v/β-1 integrin antagonists;thiazolidinediones such as rosiglitazone or troglitazone; interferon,including γ-interferon or interferon targeted to CNV by use of dextranand metal coordination; pigment epithelium derived factor (PEDF);endostatin; angiostatin; tumistatin; canstatin; anecortave acetate;acetonide; triamcinolone; tetrathiomolybdate; RNA silencing or RNAinterference (RNAi) of angiogenic factors, including ribozymes thattarget VEGF expression; Accutane™ (13-cis retinoic acid); ACEinhibitors, including but not limited to quinopril, captopril, andperindozril; inhibitors of mTOR (mammalian target of rapamycin);3-aminothalidomide; pentoxifylline; 2-methoxyestradiol; colchicines;AMG-1470; cyclooxygenase inhibitors such as nepafenac, rofecoxib,diclofenac, rofecoxib, NS398, celecoxib, vioxx, and(E)-2-alkyl-2(4-methanesulfonylphenyl)-1-phenylethene; t-RNA synthasemodulator; metalloprotease 13 inhibitor; acetylcholinesterase inhibitor;potassium channel blockers; endorepellin; purine analog of6-thioguanine; cyclic peroxide ANO-2; (recombinant) arginine deiminase;epigallocatechin-3-gallate; cerivastatin; analogues of suramin; VEGFtrap molecules; inhibitors of hepatocyte growth factor (antibodies tothe growth factor or its receptors, small molecular inhibitors of thec-met tyrosine kinase, truncated versions of HGF e.g. NK4); apoptosisinhibiting agents; Visudyne™, snET2 and other photo sensitizers withphotodynamic therapy (PDT); and laser photocoagulation.

Diseases and Conditions that May be Treated, Prevented, Inhibited, OnsetDelayed, or Regression Caused

Herein are described diseases and conditions that may be treated,prevented, inhibited, onset delayed, or regression caused using thetherapeutic agents and the formulations, liquid formulations, andmethods described herein. In some variations, the diseases or conditionsare treated using the therapeutic agents and the formulations, liquidformulations, and methods described herein. Unless the context indicatesotherwise, it is envisioned that the subjects on whom all of the methodsof treatment may be performed include, but are not limited to, humansubjects.

Generally, any diseases or condition of the eye susceptible totreatment, prevention, inhibition, delaying the onset of, or causing theregression of using the therapeutic agents and the formulations, liquidformulations and methods described herein may be treated, prevented,inhibited, onset delayed, or regression caused treated or prevented.Examples of diseases or conditions of the eye include, but are notlimited to, diseases or conditions associated with neovascularizationincluding retinal and/or choroidal neovascularization.

Diseases or conditions associated with retinal and/or choroidalneovascularization that can be treated, prevented inhibited, have onsetdelayed, or be caused to regress using the formulations, liquidformulations, and methods described herein include, but are not limitedto, diabetic retinopathy, macular degeneration, wet and dry AMD,retinopathy of prematurity (retrolental fibroplasia), infections causinga retinitis or choroiditis, presumed ocular histoplasmosis, myopicdegeneration, angioid streaks, and ocular trauma. Other non-limitingexamples of diseases and conditions of the eye that may be treated,prevented inhibited, have onset delayed, or be caused to regress usingthe formulations, liquid formulations, and methods described hereininclude, but are not limited to, pseudoxanthoma elasticum, veinocclusion, artery occlusion, carotid obstructive disease, Sickle Cellanemia, Eales disease, myopia, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma, polypoidal choroidalvasculopathy, post-laser complications, complications of idiopathiccentral serous chorioretinopathy, complications of choroidalinflammatory conditions, rubeosis, diseases associated with rubeosis(neovascularization of the angle), neovascular glaucoma, uveitis andchronic uveitis, macular edema, proliferative retinopathies and diseasesor conditions caused by the abnormal proliferation of fibrovascular orfibrous tissue, including all forms of proliferative vitreoretinopathy(including post-operative proliferative vitreoretinopathy), whether ornot associated with diabetes.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to prevent or delay onset of a disease orcondition of the eye where the subject, including but not limited to ahuman subject, is at heightened risk of developing the disease orcondition of the eye. A subject with a heightened risk of developing adisease or condition is a subject with one or more indications that thedisease or condition is likely to develop in the particular subject. Insome variations the subject with a heightened risk of developing wet AMDis a subject with dry AMD in at least one eye. In some variations thesubject with a heightened risk of developing wet AMD in a fellow eye isa subject with wet AMD in the other eye. In some variations, theformulations and pharmaceutical formulations described herein are usedto prevent or delay onset of CNV in a subject at heightened risk ofdeveloping CNV, including but not limited to prevention or delayingonset of CNV in the fellow eye of a subject, including but not limitedto a human subject with AMD in one eye. In some variations, theformulations and pharmaceutical formulations described herein are usedto prevent or delay onset of CNV in the fellow eye of a subject with wetAMD in one eye. In some variations, the formulations and pharmaceuticalformulations comprise a limus compound, including but not limited torapamycin. In some variations the formulations and pharmaceuticalformulations are administered periocularly, including without limitationsubconjunctivally, to a human subject with vision of 20/40 or better. Insome variations, the formulations and pharmaceutical formulations areadministered periocularly, including without limitationsubconjunctivally, to the eye of a human subject where the eye to whichthe formulation is administered has vision of 20/40 or better.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to treat, prevent, or delay onset of AMD. Insome variations, the formulations and pharmaceutical formulationsdescribed herein are used to treat, prevent, or delay onset of dry AMD.In some variations, subjects including but not limited to human subjectswith non-central geographic atrophy are administered a formulation orpharmaceutical formulations described herein to treat, prevent, or delayonset of central geographic atrophy. In some variations, theformulations and pharmaceutical formulations comprise a limus compound,including but not limited to rapamycin. In some variations theformulations and pharmaceutical formulations are administeredperiocularly, including without limitation subconjunctivally, to a humansubject with vision of 20/40 or better. In some variations, theformulations and pharmaceutical formulations described herein areadministered and the subject, including but not limited to a humansubject is also treated with a second therapy for treating the diseaseor disorder. In some variations, the formulations and pharmaceuticalformulations described herein are used to treat, prevent, or delay onsetof wet or dry AMD and the subject, including but not limited to a humansubject is also treated with laser therapy such as photodynamic lasertherapy, either before, during, or after treatment with the formulationsor pharmaceutical formulations described herein.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to treat one or more of uveitis, allergicconjunctivitis, macular edema, glaucoma, or dry eye.

In some variations, a formulations or pharmaceutical formulationcomprises a limus compound such as rapamycin, and is administered totreat, prevent, or delay onset of dry eye. In some variations, aformulations or pharmaceutical formulation comprises a limus compoundsuch as rapamycin, and is administered to treat, prevent, or delay onsetof allergic conjunctivitis.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to treat glaucoma. In some variations, theformulations and pharmaceutical formulations described herein fortreating glaucoma comprise a limus compound such as rapamycin, and areused as a surgical adjuvant to prevent, reduce or delay surgicalcomplications. In some variations, the formulations and pharmaceuticalformulations described herein for treating glaucoma comprise a limuscompound such as rapamycin, and are used to improve or prolong surgicalimplant success. In some variations, the formulations and pharmaceuticalformulations described herein for treating glaucoma comprise a limuscompound such as rapamycin, and are used to improve or prolong successof an argon laser trabeculectomy or other glaucoma-related surgery. Insome variations, the formulations and pharmaceutical formulationsdescribed herein have a neuroprotective effect and are used to treatglaucoma.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to treat retinitis pigmentosa. In somevariations, the formulations and pharmaceutical formulations describedherein for treating glaucoma comprise a limus compound such asrapamycin, and are used to treat, prevent, or delay onset of retinitispigmentosa. In some variations, the formulations and pharmaceuticalformulations described herein have a neuroprotective effect and are usedto treat retinitis pigmentosa.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to treat one or more of central retinal veinocclusive diseases (CRVO), branch retinal venous occlusion (BRVO),retinal vascular diseases and conditions, macular edema, diabeticmacular edema, iris neovascularization, diabetic retinopathy, or cornealgraft rejection. In some variations, a formulations or pharmaceuticalformulation comprises a limus compound such as rapamycin, and isadministered to treat, prevent, or delay onset of one or more of thesediseases or conditions. In some variations the formulations andpharmaceutical formulations are administered subconjunctivally to an eyewith vision of 20/40 or better.

When used to treat, prevent, inhibit, delay the onset of, or causeregressions of uveitis, the formulations and pharmaceutical formulationsdescribed herein may be administered by a variety of routes as is knownin the art, including but not limited to by ocular or oraladministration. Other routes of administration are known and are routinein the art. In some variations, the formulations described hereincomprise rapamycin and are used to treat uveitis.

One disease that may be treated, prevented, inhibited, have onsetdelayed, or be caused to regress using the formulation, liquidformulations and methods described herein is the wet form of AMD. Insome variations wet AMD is treated using the formulations, liquidformulations and methods described herein. The wet form of AMD ischaracterized by blood vessels growing from their normal location in thechoroid into an undesirable position under the retina. Leakage andbleeding from these new blood vessels results in vision loss andpossibly blindness.

The formulations, liquid formulations, and methods described herein mayalso be used to prevent or slow the transition from the dry form of AMD(wherein the retinal pigment epithelium or RPE degenerates and leads tophotoreceptor cell death and the formation of yellow deposits calleddrusen under the retina) to the wet form of AMD.

“Macular degeneration” is characterized by the excessive buildup offibrous deposits in the macula and retina and the atrophy of the retinalpigment epithelium. As used herein, an eye “afflicted” with maculardegeneration is understood to mean that the eye exhibits at least onedetectable physical characteristic associated with the disease ofmacular degeneration. The administration of rapamycin appears to limitand regress angiogenesis, such as choroidal neovascularization inage-related macular degeneration (AMD), which may occur withouttreatment. As used herein, the term “angiogenesis” means the generationof new blood vessels (“neovascularization”) into a tissue or organ. An“angiogenesis-mediated disease or condition” of the eye or retina is onein which new blood vessels are generated in a pathogenic manner in theeye or retina, resulting in dimunition or loss of vision or otherproblem, e.g., choroidal neovascularization associated with AMD.

The formulations and liquid formulations described herein, including butnot limited to rapamycin-containing formulations and liquidformulations, may also be used to treat, prevent, inhibit, delay theonset of, or cause regression of various immune-related diseases andconditions, including but not limited to organ transplant rejection in ahost, graft vs. host disease, autoimmune diseases, diseases ofinflammation, hyperproliferative vascular disorders, solid tumors, andfungal infections. In some variations, the formulations and liquidformulations described herein, including but not limited torapamycin-containing formulations and liquid formulations, are used totreat various immune-related diseases and conditions, including but notlimited to organ transplant rejection in a host, graft vs. host disease,autoimmune diseases, diseases of inflammation, hyperproliferativevascular disorders, solid tumors, and fungal infections. Theformulations and liquid formulations described herein, including but notlimited to rapamycin-containing formulations and liquid formulations,may be used as immunosuppressants. The formulations and liquidformulations described herein, including but not limited torapamycin-containing formulations and liquid formulations, may be usedto treat, prevent, inhibit, or delay the onset of rejection oftransplanted organs or tissues including but not limited to transplantedheart, liver, kidney, spleen, lung, small bowel, pancreas, and bonemarrow. In some variations, the formulations and liquid formulationsdescribed herein are used to treat the onset of rejection oftransplanted organs or tissues including but not limited to transplantedheart, liver, kidney, spleen, lung, small bowel, pancreas, and bonemarrow. When used to treat, prevent, inhibit, delay the onset of, orcause regressions of immune-related diseases, including but not limitedto transplant rejection, the formulations and liquid formulationsdescribed herein may be administered by a variety of routes as is knownin the art, including but not limited to by oral administration.

Systemic administration may be achieved by oral administration of theliquid formulation. Other systemic routes of administration are knownand are routine in the art. Some examples thereof are listed in theDetailed Description section.

As used herein, to “inhibit” a disease or condition by administration ofa therapeutic agent means that the progress of at least one detectablephysical characteristic or symptom of the disease or condition is slowedor stopped following administration of the therapeutic agent as comparedto the progress of the disease or condition without administration ofthe therapeutic agent.

As used herein, to “prevent” a disease or condition by administration ofa therapeutic agent means that the detectable physical characteristicsor symptom of the disease or condition do not develop followingadministration of the therapeutic agent.

As used herein, to “delay onset of” a disease or condition byadministration of a therapeutic agent means that at least one detectablephysical characteristic or symptom of the disease or condition developslater in time following administration of the therapeutic agent ascompared to the progress of the disease or condition withoutadministration of the therapeutic agent.

As used herein, to “treat” a disease or condition by administration of atherapeutic agent means that the progress of at least one detectablephysical characteristic or symptom of the disease or condition isslowed, stopped, or reversed following administration of the therapeuticagent as compared to the progress of the disease or condition withoutadministration of the therapeutic agent.

As used herein, to “cause regression of” a disease or condition byadministration of a therapeutic agent means that the progress of atleast one detectable physical characteristic or symptom of the diseaseor condition is reversed to some extent following administration of thetherapeutic agent.

A subject, including but not limited to a human subject, having apredisposition for or in need of prevention may be identified by theskilled practitioner by established methods and criteria in the fieldgiven the teachings herein. The skilled practitioner may also readilydiagnose individuals as in need of inhibition or treatment based uponestablished criteria in the field for identifying angiogenesis and/orneovascularization given the teachings herein.

As used herein, a “subject” is generally any animal that may benefitfrom administration of the therapeutic agents described herein. In somevariations the therapeutic agents are administered to a mammaliansubject. In some variations the therapeutic agents are administered to ahuman subject. In some variations the therapeutic agents may beadministered to a veterinary animal subject. In some variations thetherapeutic agents may be administered to a model experimental animalsubject.

Other diseases and conditions that may be treated, prevented, inhibited,have the onset delayed, or be caused to regress using the methodsdescribed herein include those disclosed in the following patents andpublications, the contents of each of which is incorporated herein inits entirety: PCT publication WO 2004/027027, titled Method ofinhibiting choroidal neovascularization, assigned to Trustees of theUniversity of Pennsylvania; U.S. Pat. No. 5,387,589, titled Method ofTreating Ocular Inflammation, assigned to University of LouisvilleResearch Foundation; U.S. Pat. No. 6,376,517, titled Pipecolic acidderivatives for vision and memory disorders, assigned to GPI NILHoldings, Inc; PCT publication WO 2004/028477, titled Method subretinaladministration of therapeutics including steroids: method for localizingpharmadynamic action at the choroid and retina; and related methods fortreatment and or prevention of retinal diseases, assigned to Innorx,Inc; U.S. Pat. No. 6,416,777, titled Ophthalmic drug delivery device,assigned to Alcon Universal Ltd; U.S. Pat. No. 6,713,081, titled Oculartherapeutic agent delivery device and methods for making and using suchdevices, assigned to Department of Health and Human Services; U.S. Pat.No. 5,536,729, titled Rapamycin Formulations for Oral Administration,assigned to American Home Products Corp., and U.S. application Ser. Nos.60/503,840 and 10/945,682.

Liquid Formulations

The liquid formulations described herein contain a therapeutic agent andmay generally be any liquid formulation, including but not limited tosolutions, suspensions, and emulsions. In some variations the liquidformulations form a non-dispersed mass relative to a surrounding mediumwhen placed in the vitreous of a rabbit eye.

When a certain volume of a liquid formulation is administered, it isunderstood that there is some imprecision in the accuracy of variousdevices that may be used to administer the liquid formulation. Where acertain volume is specified, it is understood that this is the targetvolume. However, certain devices such as insulin syringes are inaccurateto greater than 10%, and sometimes inaccurate to greater than 20% ormore. Hamilton HPLC type syringes are generally considered precise towithin 10%, and are recommended for volumes below 10 μl are to beinjected.

In some variations, a volume of a liquid formulation described herein isadministered to the vitreous of a rabbit eye or a subject's, includingbut not limiting a human subject's eye that is less than about 500 μl,less than about 400 μl, less than about 300 μl, less than about 200 μl,less than about 100 μl, less than about 90 μl, less than about 80 μl,less than about 70 μl, less than about 60 μl, less than about 50 μl,less than about 40 μl, less than about 30 μl, less than about 20 al,less than about 10 μl, less than about 5 μl, less than about 3 μl, orless than about 1 μl. In some variations, a volume of a liquidformulation described herein is administered to the vitreous of a rabbiteye or subject's, including but not limited to a human subject's eyethat is less than about 20 μl. In some variations, a volume of a liquidformulation described herein is administered to the vitreous that isless than about 10 μl. In some variations, a volume of a liquidformulation described herein is administered to the vitreous of a rabbiteye or a subject's, including but not limited to a human subject's eyethat is between about 0.1 μl and about 200 μl, between about 50 μl andabout 200 μl, between about 50 μl and about 150 μl, between about 0.1 μland about 100 μl, between about 0.1 μl and about 50 μl, between about 1μl and about 40 al, between about 1 μl and about 30 μl, between about 1μl and about 20 μl, between about 1 μl and about 10 μl, or between about1 μl and about 5 μl. In some variations, a volume of a liquidformulation described herein is administered to the vitreous of a rabbiteye or a subject's, including but not limited to a human subject's eyethat is between about 1 μl and about 10 μl. In some variations, a volumeof a liquid formulation described herein is administered to the vitreousof a rabbit eye or a subject's, including but not limited to a humansubject's eye that is between about 1 μl and about 5 μl. In somevariations, a volume of a liquid formulation described herein isadministered to the vitreous of a rabbit eye or a subject's eye that isbetween about 1 μl and about 5 μl. In some variations, a volume of aliquid formulation described herein is administered to the vitreous of arabbit eye or a subject's, including but not limited to a humansubject's eye that is between about 0.1 μl and about 200 μl.

In some variations, a total volume of a liquid formulation describedherein is subconjunctivally administered to a rabbit eye or a subject's,including but not limited to a human subject's eye that is less thanabout 1000 μl, less than about 900 μl, less than about 800 al, less thanabout 700 μl, less than about 600 μl, less than about 500 μl, less thanabout 400 μl, less than about 300 μl, less than about 200 μl, less thanabout 100 μl, less than about 90 μl, less than about 80 μl, less thanabout 70 μl, less than about 60 μl, less than about 50 μl, less thanabout 40 μl, less than about 30 μl, less than about 20 μl, less thanabout 10 μl, less than about 5 al, less than about 3 μl, or less thanabout 1 μl. In some variations, a volume of a liquid formulationdescribed herein is subconjunctivally administered to a rabbit eye or asubject's, including but not limited to a human subject's eye that isless than about 20 μl. In some variations, a volume of a liquidformulation described herein is subconjunctivally administered to arabbit eye or a subject's, including but not limited to a humansubject's eye that is less than about 10 μl. In some variations, avolume of a liquid formulation described herein is subconjunctivallyadministered to a rabbit eye or a subject's, including but not limitedto a human subject's eye that is between about 0.1 μl and about 200 μl,between about 50 μl and about 200 μl, between about 200 μl and about 300μl, between about 300 μl and about 400 μl, between about 400 μl andabout 500 μl, between about 600 μl and about 700 μl, between about 700μl and about 800 μl, between about 800 μl and about 900 μl, betweenabout 900 μl and about 1000 μl, between about 50 μl and about 150 μl,between about 0.141 and about 100 μl, between about 0.1 μl and about 50μl, between about 1 μl and about 40 μl, between about 1 μl and about 30μl, between about 1 μl and about 20 μl, between about 1 μl and about 10μl, or between about 1 μl and about 5 μl. In some variations, a volumeof a liquid formulation described herein is subconjunctivallyadministered to a rabbit eye or a subject's, including but not limitedto a human subject's eye that is between about 1 μl and about 10 μl. Insome variations, a volume of a liquid formulation described herein issubconjunctivally administered to a rabbit eye or a subject's, includingbut not limited to a human subject's eye that is between about 1 μl andabout 5 μl. In some variations, a volume of a liquid formulationdescribed herein is administered to subconjunctivally administered to arabbit eye or a subject's, including but not limited to a humansubject's eye that is between about 1 μl and about 5 μl. In somevariations, a volume of a liquid formulation described herein isadministered to subconjunctivally administered to a rabbit eye or asubject's, including but not limited to a human subject's eye that isbetween about 0.1 μl and about 200 μl.

In some variations the liquid formulations described herein areadministered in multiple subconjunctival locations within a period oftime, including without limitation within an hour of one another.Without being bound by theory, it is thought that such multipleadministrations, such as multiple injections, allow for a greater totaldose to be administered subconjunctivally than a single dose due to apotentially limited ability of the local ocular tissues to absorb largervolumes.

One liquid formulation described herein is an in situ gellingformulation. In situ gelling formulations, as described herein, comprisea therapeutic agent and a plurality of polymers which give a formulationthat forms a gel or a gel-like substance when placed in an aqueousmedium, including but not limited to an aqueous medium of the eye.

In some variations of the liquid formulations described herein, thetherapeutic agent is a solution or suspension of rapamycin in a liquidmedium. Liquid media include but are not limited to solvents, includingbut not limited to those in the Solubilization of Therapeutic Agentssection.

The liquid formulations described herein may comprise a solubilizingagent component. In some variations the solubilizing agent component isa surfactant. Note that there is some overlap between components thatmay be solvents and solubilizing agents, and therefore the samecomponent may in some systems be used as either a solvent or asolubilizing agent. A liquid formulation that comprises a therapeuticagent and a component that may be considered either a solvent or asolubilizing agent or surfactant will be considered a solvent if it isplaying the role of a solvent; if the component is not playing the roleof the solvent, the component may be considered a solubilizing agent orsurfactant.

Liquid formulations may optionally further comprise stabilizers,excipients, gelling agents, adjuvants, antioxidants, and/or othercomponents as described herein.

In some variations all components in the liquid formulation, other thanthe therapeutic agent, are liquid at room temperature.

In some variations, the liquid formulation comprises a release modifyingagent. In some variations, the release modifying agent is a film-formingpolymer component. The film-forming polymer component may comprise oneor more film-forming polymers. Any film-forming polymer may be used inthe excipient component. In some variations, the film-forming polymercomponent comprises a water insoluble film forming polymer. In somevariations, the release modifying agent component comprises an acrylicpolymer, including but not limited to polymethacrylate, including butnot limited to Eudragit RL.

Described herein are compositions and liquid formulations for deliveryof the therapeutic agents described in the Therapeutic Agents section.Delivery of therapeutic agents using the compositions and liquidformulations described herein may be used to treat, prevent, inhibit,delay the onset of, or cause the regression of the diseases andconditions described in the Diseases and Conditions section. Thecompositions and liquid formulations described herein may comprise anyof the therapeutic agents described in the Therapeutic Agents section,including but not limited to rapamycin. The compositions and liquidformulations described herein may comprise one or more than onetherapeutic agent. Other compositions and liquid formulations inaddition to those explicitly described herein may be used.

When the therapeutic agent is rapamycin, the compositions and liquidformulations may be used to maintain an amount of rapamycin in thevitreous effective to treat wet AMD. In one nonlimiting example, it isbelieved that a liquid formulation delivering rapamycin to maintain aconcentration of rapamycin of about 10 pg/ml to about 2 μg/ml in thevitreous over a period of time may be used for the treatment of wet AMD.When the rapamycin is in a liquid formulation that forms a non-dispersedmass, the stated concentration of rapamycin represents the amount thatis effectively treating the disease or condition of the eye, and notmerely present in the form of the non-dispersed mass. In anothernonlimiting example, it is believed that a delivery system deliveringrapamycin to maintain a concentration of rapamycin of about 0.01 pg/mgto about 10 ng/mg in the retina choroid tissues over a period of timemay be used for treatment of wet AMD. Other therapeutically effectiveamounts of therapeutic agent are also possible, and can be readilydetermined by one of skill in the art given the teachings herein.

When the therapeutic agent is rapamycin, the compositions and liquidformulations described herein may be used to deliver a dose of rapamycinto a subject, including but not limited to a human subject or to the eyeof a subject. In one nonlimiting example, it is believed that a liquidformulation containing a dose of about 20 μg to about 4 mg may be usedfor the treatment of wet AMD.

In some variations the therapeutic agent in the liquid formulationcomprises between about 0.01 to about 30% of the total weight of thecomposition; between about 0.05 to about 15%; between about 0.1 to about10%; between about 1 to about 5%; or between about 5 to about 15%;between about 8 to about 10%; between about 0.01 to about 1%; betweenabout 0.05 to about 5%; between about 0.1 to about 0.2%; between about0.2 to about 0.3%; between about 0.3 to about 0.4%; between about 0.4 toabout 0.5%; between about 0.5 to about 0.6%; between about 0.6 to about0.7%; between about 0.7 to about 1%; between about 1 to about 5%;between about 5 to about 10%; between about 15 to about 30%, betweenabout 20 to about 30%; or between about 25 to about 30%.

Those of skill in the art, based on the teachings herein can determinewhat amount or concentration of a given therapeutic agent is equivalentto an amount or concentration of rapamycin by, for example,administering the therapeutic agent at various amounts or concentrationsto a disease model system, such as an in vivo or in vivo model system,and comparing the results in the model system relative to the results ofvarious amounts or concentrations of rapamycin. Those of skill in theart, based on the teachings herein can also determine what amount orconcentration of a given therapeutic agent is equivalent to an amount orconcentration of rapamycin by reviewing the scientific literature forexperiments performed comparing rapamycin to other therapeutic agents.It is understood that even the same therapeutic agent may have adifferent equivalent level of rapamycin when, for example, a differentdisease or disorder is being evaluated, or a different type offormulation is used. Nonlimiting examples of scientific references withcomparative studies of rapamycin and other therapeutic agents on oculardisease are Ohia et al., Effects of steroids and immunosuppressive drugson endotoxin-uveitis in rabbits, J. Ocul. Pharmacol. 8(4):295-307(1992); Kulkarni, Steroidal and nonsteroidal drugs in endotoxin-induceduveitis, J. Ocul. Pharmacol. 10(1):329-34 (1994); Hafizi et al.,Differential effects of rapamycin, cyclosporine A, and FK506 on humancoronary artery smooth muscle cell proliferation and signaling, VasculPharmacol. 41(4-5):167-76 (2004); and US 2005/0187241.

For example, in a model for wet AMD, if a therapeutic agent is found tobe approximately 10-fold less potent or efficacious than rapamycin inthe treatment of wet AMD, a concentration of 10 ng/ml of the therapeuticagent would be equivalent to a 1 ng/ml concentration of rapamycin. Or ifa therapeutic agent is found to be approximately 10-fold less potent orefficacious than rapamycin in the treatment of wet AMD, a 10-fold amountof the therapeutic agent would be administered relative to the amount ofrapamycin.

The solvent component may comprise, for instance, between about 0.01 toabout 99.9% of the total weight of the composition; between about 0.1 toabout 99%; between about 25 to about 55%; between about 30 to about 50%;or between about 35 to about 45%; between about 0.1 to about 10%;between about 10 to about 20%; between about 20 to about 30%; betweenabout 30 to about 40%; between about 40 to about 45%; between about 40to about 45%; between about 45 to about 50%; between about 50 to about60%; between about 50 to about 70%; between about 70 to about 80%;between about 80 to about 90%; or between about 90 to about 100%.

The solubilizing agent component may comprise, for instance, betweenabout 0.01 to about 30% of the total weight of the composition; betweenabout 0.1 to about 20%; between about 2.5 to about 15%; between about 10to about 15%; or between about 5 to about 10%; between about 8 to about12%; between about 10 to about 20%; between about 20 to about 30%.

In some variations, the liquid formulations described herein have aviscosity of between 40% and 120% centipoise. In some variations theliquid formulations described herein have a viscosity of between 60% and80% centipoise.

In some variations the liquid formulations described herein comprise atherapeutic agent and a solvent component. The solvent component maycomprise a single solvent or a combination of solvents. The therapeuticagent component may comprise a single therapeutic agent or a combinationof therapeutic agents. In some variations, the solvent is glycerin,dimethylsulfoxide, N-methylpyrrolidone, dimethyl acetamide (DMA),dimethyl formamide, glycerol formal, ethoxy diglycol, triethylene glycoldimethyl ether, triacetin, diacetin, corn oil, acetyl triethyl citrate(ATC), ethyl lactate, polyglycolated capryl glyceride, γ butyrolactone,dimethyl isosorbide, benzyl alcohol, ethanol, isopropyl alcohol,polyethylene glycol of various molecular weights, including but notlimited to PEG 300 and PEG 400, or propylene glycol, or a mixture of oneor more thereof.

In some variations the liquid formulations described herein aresolutions, and comprise a therapeutic agent and a solvent component. Insome variations the solvent component comprises ethanol. In somevariations the solvent component comprises ethanol and a polyethyleneglycol, including but not limited to a liquid polyethylene glycol,including but not limited to one or more of PEG 300 or PEG 400.

In some variations the liquid formulations described herein contain nogreater than about 250 μl of polyethylene glycol. In some variations theliquid formulations described herein contain no greater than about 250μl, no greater than about 200 μl, no greater than about 150 μl, nogreater than about 125 μl, no greater than about 100 μl, no greater thanabout 75 μl, no greater than about 50 μl, no greater than about 25 μl,no greater than about 20 μl, no greater than about 15 μl, no greaterthan about 10 μl, no greater than about 7.5 μl, no greater than about 5μl, no greater than about 2.5 μl, no greater than about 1.0 μl, or nogreater than about 0.5 μl of polyethylene glycol. Formulationscontaining polyethylene glycol may contain, for example, PEG 300 or PEG400.

In some variations, the liquid formulations described herein aresuspensions, and comprise a therapeutic agent and a diluent component.In some variations, the diluent component comprises one or morecomponents listed herein as solvents or solubilizing agents, wherein theresulting mixture is a suspension.

In some variations the liquid formulation is partly a solution andpartly a suspension.

In some variations the liquid formulation is an in situ gellingformulation, and comprises a therapeutic agent and a polymer component,wherein the polymer component may comprise a plurality of polymers. Insome variations, the liquid formulation comprises a polymethacrylatepolymer. In some variations, the liquid formulation comprises apolyvinylpyrrolidone polymer.

Some variations of liquid formulations include a therapeutic agent oragents such as but not limited to rapamycin between about 0.01% andabout 20% by weight of the total, a solvent between about 5% and about15% by weight of the total, a solubilizing agent including but notlimited to a surfactant between about 5% and about 15% by weight of thetotal, with water as the primary remaining component. In some variationsthe formulations further comprise stabilizing agents, excipients,adjuvants, or antioxidants, between about 0 and about 40% by weight ofthe total.

In some variations, a liquid formulation comprises up to about 5%therapeutic agent, including but not limited to rapamycin, per weight ofthe total; and up to about 99.9% of a solvent component, by weight ofthe total. In some variations the liquid formulation comprises up toabout 5% therapeutic agent, including but not limited to rapamycin, perweight of the total; and up to about 99.9% of a diluent component.

In some variations, a liquid formulation may comprise up to about 5%therapeutic agent, including but not limited to rapamycin, per weight ofthe total; up to about 10% solvent by weight of the total; and up toabout 85% of a solubilizing component, by weight of the total. In somevariations the solubilizing component is an aqueous solution of asurfactant.

A plurality of polymers component may comprise, for instance, betweenabout 0.01 to about 30% of the total weight of the composition; betweenabout 0.1 to about 20%; between about 2.5 to about 15%; between about 10to about 15%; between about 3 to about 5%; between about 5 to about 10%;between about 8 to about 12%; between about 10 to about 20%; or betweenabout 20 to about 30%.

Some variations of liquid formulations includes a therapeutic agent oragents such as but not limited to rapamycin between about 0.01% andabout 20% by weight of the total, a solvent component between about 60%and about 98% by weight of the total, and a plurality of polymers, whosecombined percentage is between about 0.1% and about 15% by weight of thetotal. In some variations the formulations further comprise stabilizingagents, excipients, adjuvants, or antioxidants, between about 0 andabout 40% by weight of the total.

In some variations, a liquid formulation may comprise about 4%therapeutic agent, including but not limited to rapamycin, per weight ofthe total; about 91% solvent by weight of the total; and about 5%polymeric component, per weight of the total.

Some examples and variations of liquid formulations described hereinwere prepared and are listed in Table 1. Depending on their type, thelisted formulations are denoted one or more of solutions (“S”),suspensions (“SP”), emulsions (“E”) or in situ gelling (“ISG”). Medianparticle size is listed for some of the suspensions. As describedherein, some liquid formulations form a non-dispersed mass after, forexample, injection into an aqueous environment such as the vitreous ofan eye. For those formulations injected into the vitreous of a rabbiteye, the right-hand column of Table 1 indicates whether or not anon-dispersed mass (NDM) formed after a specified volume was injectedinto the vitreous of the rabbit eye.

The following references, each of which is incorporated herein byreference in its entirety, show one or more formulations, including butnot limited to rapamycin formulations, and which describe use ofrapamycin at various doses and other therapeutic agents for treatingvarious diseases or conditions: U.S. 60/651,790, filed Feb. 9, 2005,titled FORMULATIONS FOR OCULAR TREATMENT, attorney docket number57796-30002.00; U.S. 60/664,040, filed Feb. 9, 2005, attorney docketnumber 57796-30004.00, titled LIQUID FORMULATIONS FOR TREATMENT OFDISEASES OR CONDITIONS; U.S. 60/664,119, filed Mar. 21, 2005, attorneydocket number 57796-30005.00, titled DRUG DELIVERY SYSTEMS FOR TREATMENTOF DISEASES OR CONDITIONS; U.S. 60/664,306, filed Mar. 21, 2005,attorney docket number 57796-30006.00 titled IN SITU GELLINGFORMULATIONS AND LIQUID FORMULATIONS FOR TREATMENT OF DISEASES ORCONDITIONS; U.S. Ser. No. 11/351,844, filed Feb. 9, 2006, titledFORMULATIONS FOR OCULAR TREATMENT, attorney docket number57796-20002.00; US 2005/0187241, and US 2005/0064010.

Liquid Formulations which Form a Non-Dispersed Mass

One class of liquid formulations described herein forms a non-dispersedmass when placed in an aqueous medium. As used herein, a “non-dispersedmass” refers to the structure formed or shape assumed when the liquidformulation is placed into an environment, relative to the environmentin which it is placed. Generally, a non-dispersed mass of a liquidformulation is anything other than a homogeneous distribution of theliquid formulation in the surrounding medium. The non-dispersed massmay, for instance, be indicated by visually inspecting the administeredliquid formulation and characterizing its appearance relative to thesurrounding medium.

In some variations, the aqueous medium is water. In some variations, thewater is deionized, distilled, sterile, or tap water, including but notlimited to tap water available at the place of business of MacuSight inUnion City, Calif.

In some variations, the aqueous medium is an aqueous medium of asubject. In some variations the aqueous medium is an aqueous medium ofthe eye of a subject, including but not limited to the vitreous of aneye of a subject. In some variations the subject is a human subject. Insome variations the subject is a rabbit.

In some variations the liquid formulation forms a non-dispersed masswhen exposed to a certain temperature or range of temperatures,including but not limited to about room temperature, about ambienttemperature, about 30° C., about 37° C., or about the temperature of theaqueous medium of the subject.

In some variations the liquid formulation forms a non-dispersed masswhen exposed to a certain pH or range of pH, including but not limitedto a pH between about 6 and about 8.

In some variations, the non-dispersed mass comprises a gel or gel-likesubstance.

In some variations, the non-dispersed mass comprises a polymer matrix.In some variations, the non-dispersed mass comprises a polymer matrix inwhich a therapeutic agent is dispersed.

The liquid formulations described herein may generally be of anygeometry or shape after administration to a subject or the eye of asubject, including but not limited to a human subject. In somevariations, the non-dispersed mass is between about 0.1 and about 5 mm.In some variations, the non-dispersed mass is between about 1 and about3 mm. The non-dispersed mass-forming liquid formulations may, forinstance, appear as a compact spherical mass when administered to thevitreous. In some instances, the liquid formulation may appear as anon-dispersed mass relative to the surrounding medium, wherein thenon-dispersed mass is less clearly defined and the geometry is moreamorphous than spherical.

The non-dispersed mass-forming liquid formulations described herein mayform a non-dispersed mass immediately upon placement in the medium orthe non-dispersed mass may form some period of time after placement ofthe liquid formulation. In some variations the non-dispersed mass formsover the course of about 1, about 2, about 3, about 4, about 5, about 6,or about 7 days. In some variations the non-dispersed mass forms overthe course of about 1 week, about 2 weeks, or about 3 weeks.

In some variations, the liquid formulations described herein that form anon-dispersed mass appear as a milky or whitish colored semi-contiguousor semi-solid non-dispersed mass relative to the medium in which it isplaced.

One liquid formulation described herein forms a non-dispersed mass whichhas the form of a solid depot when the formulation is injected into anyor all of water, the vitreous of a rabbit eye, or between the sclera andthe conjunctiva of a rabbit eye. One liquid formulation described hereinforms a non-dispersed mass which has the form of a semi-solid when theformulation is injected into any or all of water, the vitreous of arabbit eye, or between the sclera and the conjunctiva of a rabbit eye.One liquid formulation described herein forms a non-dispersed mass whichhas the form of a polymeric matrix when the formulation is injected intoany or all of water, the vitreous of a rabbit eye, or between the scleraand the conjunctiva of a rabbit eye. One liquid formulation describedherein forms a non-dispersed mass which has the form of a gel, ahydrogel, or a gel-like substance when the formulation is injected intoany or all of water, the vitreous of a rabbit eye, or between the scleraand the conjunctiva of a rabbit eye.

In some variations described herein the liquid formulation forms anon-dispersed mass relative to a surrounding medium where thesurrounding medium is aqueous. An “aqueous medium” or “aqueousenvironment” is one that contains at least about 50% water. Examples ofaqueous media include but are not limited to water, the vitreous,extracellular fluid, conjunctiva, sclera, between the sclera and theconjunctiva, aqueous humor, gastric fluid, and any tissue or body fluidcomprised of at least about 50% of water. Aqueous media include but arenot limited to gel structures, including but not limited to those of theconjunctiva and sclera.

In some variations, the liquid formulations described herein form anon-dispersed mass when a test volume of the liquid formulation isplaced in the vitreous of a rabbit eye. In some variations the testvolume administered to a rabbit eye, and the test volume is equal to thevolume of the liquid formulation administered to a subject's, includingbut not limited to a human subject's eye.

In some variations, the test volume administered to a rabbit eye isequal to the volume administered to the subject's eye multiplied by ascale factor, and the scale factor is equal to the average volume of arabbit eye divided by the average volume of a subject eye. The “averagevolume” of an eye, as used herein, refers to the average volume of aneye of a member of similar age of the species under considerationgenerally, as opposed to the average volume of any particularindividual's eye.

In some variations, the test volume administered to the rabbit eye isbetween about 10 μl and about 50 μl. In some variations, the test volumeadministered to the rabbit eye is between about 1 μl and about 30 μl. Insome variations, the test volume administered to the rabbit eye isbetween about 50 μl and about 100 μl. In some variations, the testvolume administered to the rabbit eye is between about 25 μl and about75 μl. In some variations, the test volume administered to the rabbiteye is about 30 μl.

In some variations, the liquid formulation that forms a non-dispersedmass when placed in the medium may comprises a therapeutic agent oragents with a concentration of between about 0.01% and about 10% byweight of the total, and a solvent between about 10% and about 99% byweight of the total. In some variations the formulation furthercomprises a solubilizing agent including but not limited to asurfactant. In some variations the liquid formulation further comprisesa stabilizing agent, excipient, adjuvant, or antioxidant, etc., betweenabout 0 and about 40% by weight of the total. In some variations, thetherapeutic agent is about 5% by weight of the total, and the solventcomponent is about 95% by weight of the total.

Whether a liquid formulation exhibits a non-dispersed mass relative to asurrounding medium when present in a subject, including but not limitedto a human subject or the eye of a subject may be determined by, forinstance, mixing a therapeutic agent with a solvent, administering it tothe vitreous of an eye of a subject, including but not limited to ahuman subject, and comparing the liquid formulation to the surroundingmedium.

One liquid formulation that may be used for treating, preventing,inhibiting, delaying the onset of, or causing the regression of thediseases and conditions of a subject, including but not limited to ahuman subject, is a liquid formulation that forms a non-dispersed masswhen placed into the vitreous of a rabbit eye. When used for treating,preventing, inhibiting, delaying the onset of, or causing the regressionof the disease or condition of the subject, the liquid formulation isadministered to the subject. The liquid formulation may or may not forma non-dispersed mass in the subject. One liquid formulation describedherein forms a non-dispersed mass when administered to a subject andforms a non-dispersed mass when administered to a rabbit eye.

Without being bound by theory, it is believed that the low solubility ofrapamycin in the vitreous contributes to the formation of anon-dispersed mass by some rapamycin-containing liquid formulationsdescribed herein. The vitreous is a clear gel composed almost entirelyof water (up to 99%). Without being bound by theory, it is believed thatas rapamycin in an injected formulation contacts the vitreous, therapamycin precipitates.

Without being bound by theory, factors believed to affect the formationof and geometry of a non-dispersed mass include the concentration ofrapamycin in the formulation, the viscosity of the formulation, ethanolcontent of the formulation, and the volume of injection. It is believedthat maintaining a higher local concentration of rapamycin afterinjection of the formulation favors formation of a non-dispersed mass,as opposed to a lower local concentration of rapamycin after injectionof the formulation. As volume is increased for a given dose, formationof a non-dispersed mass may become less favorable. Formation of anon-dispersed mass may become more favorable as rapamycin concentrationis increased and/or as viscosity is increased. Ethanol content affectsboth the solubility of the rapamycin in the formulation and theviscosity of the formulation.

In one comparison, 100 μl of a solution of 0.4% rapamycin, 4.0% ethanol,and 95.6% PEG 400 did not form a non-dispersed mass after injection intoa rabbit eye. In contrast, 20 μl of a solution of 2.0% rapamycin, 4.0%ethanol, and 94% PEG 400 (an equivalent dose) formed a compact sphericalnon-dispersed mass after injection into a rabbit eye.

Without being bound by theory, in the latter example, it is hypothesizedthat formation of the non-dispersed mass occurred as depicted in FIGS.1A-1C and described as follows. Upon injection, due to its viscosity theliquid formulation formed a spherical globule 100 within the vitreous110. Ethanol then diffused out of this globule, resulting in localizedprecipitation 120 of the rapamycin within the globule. Eventually, thepolyethylene glycol also diffused out of the globule to leave a solid,compact non-dispersed mass of rapamycin 130.

In some variations, the non-dispersed masses described herein consistsof at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, or at least about 95% by volume of therapeutic agentwhen injected into the vitreous of a rabbit eye.

In some variations, upon formation a non-dispersed mass comprisingrapamycin, for example, delivers the drug continuously at approximatelya constant rate for an extended period of time. Without being bound bytheory, it is believed that delivery of rapamycin from a non-dispersedmass in the vitreous depends on dissolution of the rapamycin in thevitreous, which depends in turn on clearance of the drug from thevitreous to other tissues. Without being bound by theory, this releaseprocess is believed to maintain a steady-state concentration ofrapamycin in the vitreous.

In some variations, formation of a non-dispersed mass reduces thetoxicity of the injected liquid formulation compared to an equivalentdose that did not form a non-dispersed mass. In variations in which aliquid formulation injected into the vitreous does not form anon-dispersed mass, the drug (e.g., rapamycin) appears to disperse inthe vitreous body. In some variations this may interfere with vision.

In some variations, liquid formulations that are suspensions form anon-dispersed mass upon injection into the vitreous. Formation of anon-dispersed mass from an injected suspension may become more favorableas the suspension particle size increases.

In some variations, it is believed that the liquid formulations willform a visually observable non-dispersed mass when injected into the eyeof a subject, including but not limited to a human subject.

In some variations, liquid formulations are believed to formnon-dispersed masses when injected subconjunctivally. In some variationsit is believed that when subconjunctivally administered the liquidformulation forms a depot in the scleral tissue. That is, it is believedthat the therapeutic agent is absorbed into the sclera proximate to theinjection site and forms a local concentration of drug in the sclera.

In Situ Gelling Formulations

Described herein are non-dispersed mass-forming liquid formulationswhich form a gel or gel-like substance when placed in an aqueous medium.In some variations, the non-dispersed mass comprises a gel; in somevariations the gel is a hydrogel.

An “in situ gelling formulation,” as used herein, refers to a liquidformulation which forms a gel-like non-dispersed mass when the liquidformulation is placed in an aqueous medium, including but not limited toaqueous media that are water, the vitreous of a rabbit eye, and betweenthe sclera and the conjunctiva of a rabbit eye. In some variations, anin situ gelling formulation forms a gel-like non-dispersed mass whenplaced in tap water.

In some variations, the in situ gelling formulation is a suspensionprior to placement in an aqueous medium, and forms a gel in situ uponplacement in an aqueous medium. In some variations, the in situ gellingformulation is a solution prior to placement in an aqueous medium, andforms a gel in situ upon placement in an aqueous medium. In somevariations, the in situ gelling formulation is an emulsion prior toplacement in an aqueous medium, and forms a gel in situ upon placementin an aqueous medium. In some variations a gel-like non-dispersed massforms after placement of the in situ gelling formulation into an aqueousmedium, including but not limited to any or all of water, the vitreous,or between the sclera and the conjunctiva of an eye. In some variations,the in situ gel is formed of a polymer matrix. In some variations atherapeutic agent is dispersed in the polymer matrix.

Described herein are in situ gelling formulations which may be used fortreating, preventing, inhibiting, delaying the onset of, or causing theregression of the diseases and conditions of a subject including but notlimited to a human subject. When used for treating, preventing,inhibiting, delaying the onset of, or causing the regression of thedisease or condition of the subject, the in situ gelling formulation isadministered to the subject. One liquid formulation described hereincomprises an in situ gelling formulation which forms a non-dispersedmass when administered to a subject and forms a non-dispersed mass whenadministered to a rabbit eye.

In some variations, the in situ gelling formulation comprises one ormore polymers. Described herein are various types of polymers, includingpolymers which are solvents, polymers which are solubilizing agents,polymers which are release modifying agents, polymers which arestabilizing agents, etc. In some variations, any combination of polymersis used wherein the polymers when combined with the therapeutic agentform any or all of a non-dispersed mass, a gel, a hydrogel, or polymericmatrix when placed in an aqueous medium, including but not limited toany or all of water, the vitreous, or between the sclera and theconjunctiva.

In some variations, the in situ gelling formulation delivers extendedrelease of therapeutic agents to a subject when administered to thesubject.

In some variations, the liquid formulation comprises a therapeutic agentand a plurality of polymers, wherein one of the polymers is apolymethacrylate. Polymethacrylates are known by various names and areavailable in various preparations, including but not limited topolymeric methacrylates, methacrylic acid-ethyl acrylate copolymer(1:1), methacrylic acid-ethyl acrylate copolymer (1:1) dispersion 30percent, methacrylic acid-methyl methacrylate copolymer (1:1),methacrylic acid-methyl methacrylate copolymer (1:2), acidummethacrylicum et ethylis acrylas polymerisatum 1:1, acidum methacrylicumet ethylis acrylas polymerisatum 1:1 dispersio 30 per centum, acidummethacrylicum et methylis methacrylas polymerisatum 1:1, acidummethacrylicum et methylis methacrylas polymerisatum 1:2, USPNF: ammoniomethacrylate copolymer, methacrylic acid copolymer, methacrylic acidcopolymer dispersion.

In some variations, one of the polymers is polyvinylpyrrolidone.Polyvinylpyrrolidone is known by various names and is available invarious preparations, including but not limited to povidone, povidonum,kollidon; plasdone; poly[1-(2-oxo-1-pyrrolidinyl)ethylene]; polyvidone;PVP; 1-vinyl-2-pyrrolidinone polymer, and 1-Ethenyl-2-pyrrolidinonehomopolymer.

One liquid formulation described herein comprises a therapeutic agentand a solvent component. The solvent component may comprise a singlesolvent or a combination of solvents.

In some variations, the solvent is glycerin, dimethylsulfoxide,N-methylpyrrolidone, ethanol, isopropyl alcohol, polyethylene glycol ofvarious molecular weights, including but not limited to PEG 300 and PEG400, or propylene glycol, or a mixture of one or more thereof.

In some variations, the solvent is polyethylene glycol. Polyethyleneglycol is known by various names and is available in variouspreparations, including but not limited to macrogels, macrogel 400,macrogel 1500, macrogel 4000, macrogel 6000, macrogel 20000, macrogola,breox PEG; carbowax; carbowax sentry; Hodag PEG; Lipo; Lipoxol; LutrolE; PEG; Pluriol E; polyoxyethylene glycol, andα-Hydro-ω-hydroxy-poly(oxy-1,2-ethanediyl).

Compositions and Liquid Formulations for Delivery of Therapeutic Agents

The compositions and liquid formulations described herein may be used todeliver amounts of the therapeutic agents effective for treating,preventing, inhibiting, delaying on set of, or causing the regression ofthe diseases and conditions described in the Diseases and Conditionssection. In some variations the compositions and liquid formulationsdescribed herein deliver one or more therapeutic agents over an extendedperiod of time.

An “effective amount,” which is also referred to herein as a“therapeutically effective amount,” of a therapeutic agent foradministration as described herein is that amount of the therapeuticagent that provides the therapeutic effect sought when administered tothe subject, including but not limited to a human subject. The achievingof different therapeutic effects may require different effective amountsof therapeutic agent. For example, the therapeutically effective amountof a therapeutic agent used for preventing a disease or condition may bedifferent from the therapeutically effective amount used for treating,inhibiting, delaying the onset of, or causing the regression of thedisease or condition. In addition, the therapeutically effective amountmay depend on the age, weight, and other health conditions of thesubject as is well know to those versed in the disease or conditionbeing addressed. Thus, the therapeutically effective amount may not bethe same in every subject to which the therapeutic agent isadministered.

An effective amount of a therapeutic agent for treating, preventing,inhibiting, delaying the onset of, or causing the regression of aspecific disease or condition is also referred to herein as the amountof therapeutic agent effective to treat, prevent, inhibit, delay theonset of, or cause the regression of the disease or condition.

To determine whether a level of therapeutic agent is a “therapeuticallyeffective amount” to treat, prevent, inhibit, delay on set of, or causethe regression of the diseases and conditions described in the Diseasesand Conditions section, liquid formulations may be administered inanimal models for the diseases or conditions of interest, and theeffects may be observed. In addition, dose ranging human clinical trialsmay be conducted to determine the therapeutically effective amount of atherapeutic agent.

Generally, the therapeutic agent may be formulated in any composition orliquid formulation capable of delivery of a therapeutically effectiveamount of the therapeutic agent to a subject or to the eye of a subjectfor the required delivery period. Compositions include liquidformulations.

Solubilization of Therapeutic Agents

One composition or liquid formulation that may be used is a compositionor liquid formulation in which the therapeutic agent is dissolved in asolvent component. Generally, any solvent which has the desired effectmay be used in which the therapeutic agent dissolves. In some variationsthe solvent is aqueous. In some variations the solvent is non-aqueous.An “aqueous solvent” is a solvent that contains at least about 50%water.

Generally, any concentration of solubilized therapeutic agent that hasthe desired effect can be used. The solvent component may be a singlesolvent or may be a mixture of solvents. The solvent component may be asingle solvent or may be a mixture of solvents. Solvents and types ofsolutions are well known to those versed in such drug deliverytechnologies. See for example, Remington: The Science and Practice ofPharmacy, Twentieth Edition, Lippincott Williams & Wilkins; 20th edition(Dec. 15, 2000); Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Eighth Edition, Lippincott Williams & Wilkins (August 2004);Handbook Of Pharmaceutical Excipients 2003, American PharmaceuticalAssociation, Washington, D.C., USA and Pharmaceutical Press, London, UK;and Strickley, solubilizing Excipients in Oral and InjectableFormulations, Pharmaceutical Research, Vol. 21, No. 2, February 2004.

As noted previously, some solvents may also serve as solubilizingagents.

Solvents that may be used include but are not limited to DMSO, ethanol,methanol, isopropyl alcohol; castor oil, propylene glycol, glycerin,polysorbate 80, benzyl alcohol, dimethyl acetamide (DMA), dimethylformamide (DMF), triacetin, diacetin, corn oil, acetyl triethyl citrate(ATC), ethyl lactate, glycerol formal, ethoxy diglycol (Transcutol,Gattefosse), tryethylene glycol dimethyl ether (Triglyme), dimethylisosorbide (DMI), γ-butyrolactone, N-Methyl-2-pyrrolidinone (NMP),polyethylene glycol of various molecular weights, including but notlimited to PEG 300 and PEG 400, and polyglycolated capryl glyceride(Labrasol, Gattefosse), combinations of any one or more of theforegoing, or analogs or derivatives of any one or more of theforegoing.

In some variations, the solvent is a polyethylene glycol. Polyethyleneglycol is known by various names and is available in variouspreparations, including but not limited to macrogels, macrogel 400,macrogel 1500, macrogel 4000, macrogel 6000, macrogel 20000, macrogola,breox PEG; carbowax; carbowax sentry; Hodag PEG; Lipo; Lipoxol; LutrolE; PEG; Pluriol E; polyoxyethylene glycol, andα-Hydro-ω-hydroxy-poly(oxy-1,2-ethanediyl).

In some variations the polyethylene glycol is a liquid PEG, and is oneor more of PEG 300 or PEG 400.

Other solvents include an amount of a C₆-C₂₄ fatty acid sufficient tosolubilize a therapeutic agent.

Phospholipid solvents may also be used, such as lecithin,phosphatidylcholine, or a mixture of various diglycerides of stearic,palmitic, and oleic acids, linked to the choline ester of phosphoricacid; hydrogenated soy phosphatidylcholine (HSPC),distearoylphosphatidylglycerol (DSPG),L-α-dimyristoylphosphatidylcholine (DMPC),L-α-dimyristoylphosphatidylglycerol (DMPG).

Further examples of solvents include, for example, components such asalcohols, propylene glycol, polyethylene glycol of various molecularweights, propylene glycol esters, propylene glycol esterified with fattyacids such as oleic, stearic, palmic, capric, linoleic, etc; mediumchain mono-, di-, or triglycerides, long chain fatty acids, naturallyoccurring oils, and a mixture thereof. The oily components for thesolvent system include commercially available oils as well as naturallyoccurring oils. The oils may further be vegetable oils or mineral oils.The oils can be characterized as non-surface active oils, whichtypically have no hydrophile lipophile balance value. Commerciallyavailable substances comprising medium chain triglycerides include, butare not limited to, Captex 100, Captex 300, Captex 355, Miglyol 810,Miglyol 812, Miglyol 818, Miglyol 829, and Dynacerin 660. Propyleneglycol ester compositions that are commercially available encompassCaptex 200 and Miglyol 840, and the like. The commercial product, CapmulMCM, comprises one of many possible medium chain mixtures comprisingmonoglycerides and diglycerides.

Other solvents include naturally occurring oils such as peppermint oil,and seed oils. Exemplary natural oils include oleic acid, castor oil,safflower seed oil, soybean oil, olive oil, sunflower seed oil, sesameoil, and peanut oil. Soy fatty acids may also be used. Examples of fullysaturated non-aqueous solvents include, but are not limited to, estersof medium to long chain fatty acids (such as fatty acid triglycerideswith a chain length of about C₆ to about C₂₄). Hydrogenated soybean oiland other vegetable oils may also be used. Mixtures of fatty acids maybe split from the natural oil (for example coconut oil, palm kernel oil,babassu oil, or the like) and refined. In some embodiments, medium chain(about C₈ to about C₁₂) triglycerides, such as caprilyic/caprictriglycerides derived from coconut oil or palm seed oil, may be used.Medium chain mono- and diglycerides may also be used. Other fullysaturated non-aqueous solvents include, but are not limited to,saturated coconut oil (which typically includes a mixture of lauric,myristic, palmitic, capric and caproic acids), including those soldunder the Miglyol™ trademark from Huls and bearing trade designations810, 812, 829 and 840). Also noted are the NeoBee™ products sold by DrewChemicals. Non-aqueous solvents include isopropyl myristate. Examples ofsynthetic oils include triglycerides and propylene glycol diesters ofsaturated or unsaturated fatty acids having 6 to 24 carbon atoms suchas, for example hexanoic acid, octanoic (caprylic), nonanoic(pelargonic), decanoic (capric), undecanoic, lauric, tridecanoic,tetradecanoic (myristic), pentadecanoic, hexadecanoic (palmitic),heptadecanoic, octadecanoic (stearic), nonadecanoic, heptadecanoic,eicosanoic, heneicosanoic, docosanoic and lignoceric acids, and thelike. Examples of unsaturated carboxylic acids include oleic, linoleicand linolenic acids, and the like. The non-aqueous solvent can comprisethe mono-, di- and triglyceryl esters of fatty acids or mixed glyceridesand/or propylene glycol mono- or diesters wherein at least one moleculeof glycerol has been esterified with fatty acids of varying carbon atomlength. A non-limiting example of a “non-oil” useful as a solvent ispolyethylene glycol.

Exemplary vegetable oils include cottonseed oil, corn oil, sesame oil,soybean oil, olive oil, fractionated coconut oil, peanut oil, sunfloweroil, safflower oil, almond oil, avocado oil, palm oil, palm kernel oil,babassu oil, beechnut oil, linseed oil, rape oil and the like. Mono-,di-, and triglycerides of vegetable oils, including but not limited tocorn, may also be used.

Polyvinyl pyrrolidone (PVP), cross-linked or not, may also be used as asolvent. Further solvents include but are not limited to C₆-C₂₄ fattyacids, oleic acid, Imwitor 742, Capmul, F68, F68 (Lutrol), PLURONICSincluding but not limited to PLURONICS F108, F127, and F68, Poloxamers,Jeffamines), Tetronics, F127; cyclodextrins such as α-cyclodextrin,β-cyclodextrin, hydroxypropyl-β-cyclodextrin,sulfobutylether-β-cyclodextrin (Captisol); CMC, polysorbitan 20,Cavitron, polyethylene glycol of various molecular weights including butnot limited to PEG 300 and PEG 400.

Beeswax and d-α-tocopherol (Vitamin E) may also be used as solvents.

Solvents for use in the liquid formulations can be determined by avariety of methods known in the art, including but not limited to (1)theoretically estimating their solubility parameter values and choosingthe ones that match with the therapeutic agent, using standard equationsin the field; and (2) experimentally determining the saturationsolubility of therapeutic agent in the solvents, and choosing the onesthat exhibit the desired solubility.

Solubilization of Rapamycin

Where the therapeutic agent is rapamycin, solvents that may be used formaking solutions or suspensions of rapamycin include but are not limitedto any solvent described herein, including but not limited to any one ormore of DMSO, glycerin, ethanol, methanol, isopropyl alcohol; castoroil, propylene glycol, polyvinylpropylene, glycerin, polysorbate 80,benzyl alcohol, dimethyl acetamide (DMA), dimethyl formamide (DMF),glycerol formal, ethoxy diglycol (Transcutol, Gattefosse), tryethyleneglycol dimethyl ether (Triglyme), dimethyl isosorbide (DMI),γ-butyrolactone, N-Methyl-2-pyrrolidinone (NMP), polyethylene glycol ofvarious molecular weights, including but not limited to PEG 300 and PEG400, and polyglycolated capryl glyceride (Labrasol, Gattefosse).

Further solvents include but are not limited to C₆-C₂₄ fatty acids,oleic acid, Imwitor 742, Capmul, F68, F68 (Lutrol), PLURONICS includingbut not limited to PLURONICS F108, F127, and F68, Poloxamers,Jeffamines), Tetronics, F127, beta-cyclodextrin, CMC, polysorbitan 20,Cavitron, softigen 767, captisol, and sesame oil.

Other methods that may be used to dissolve rapamycin are described inSolubilization of Rapamycin, P. Simamora et al. Int'l J. Pharma 213(2001) 25-29, the contents of which is incorporated herein in itsentirety.

As a nonlimiting example, rapamycin can be dissolved in 5% DMSO ormethanol in a balanced salt solution. The rapamycin solution can beunsaturated, a saturated or a supersaturated solution of rapamycin. Therapamycin solution can be in contact with solid rapamycin. In onenonlimiting example, rapamycin can be dissolved in a concentration of upto about 400 mg/ml. Rapamycin can also, for example, be dissolved inpropylene glycol esterified with fatty acids such as oleic, stearic,palmic, capric, linoleic, etc.

Many other solvents are possible. Those of ordinary skill in the artwill find it routine to identify solvents for rapamycin given theteachings herein.

Solubilizing Agents

Generally, any solubilizing agent or combination of solubilizing agentsmay be used in the liquid formulations described herein.

In some variations, the solubilizing agent is a surfactant orcombination of surfactants. Many surfactants are possible. Combinationsof surfactants, including combinations of various types of surfactants,may also be used. For instance, surfactants which are nonionic, anionic(i.e. soaps, sulfonates), cationic (i.e. CTAB), zwitterionic, polymericor amphoteric may be used.

Surfactants that can be used may be determined by mixing a therapeuticagent of interest with a putative solvent and a putative surfactant, andobserving the characteristics of the formulation after exposure to amedium.

Examples of surfactants include but are not limited to fatty acid estersor amides or ether analogues, or hydrophilic derivatives thereof;monoesters or diesters, or hydrophilic derivatives thereof; or mixturesthereof; monoglycerides or diglycerides, or hydrophilic derivativesthereof; or mixtures thereof; mixtures having enriched mono- or/anddiglycerides, or hydrophilic derivatives thereof; surfactants with apartially derivatized with a hydrophilic moiety; monoesters or diestersor multiple-esters of other alcohols, polyols, saccharides oroligosaccharides or polysaccharides, oxyalkylene oligomers or polymersor block polymers, or hydrophilic derivatives thereof, or the amideanalogues thereof; fatty acid derivatives of amines, polyamines,polyimines, aminoalcohols, aminosugars, hydroxyalkylamines,hydroxypolyimines, peptides, polypeptides, or the ether analoguesthereof.

Hydrophilic Lipophilic Balance (“HLB”) is an expression of the relativesimultaneous attraction of a surfactant for water and oil (or for thetwo phases of the emulsion system being considered).

Surfactants are characterized according to the balance between thehydrophilic and lipophilic portions of their molecules. Thehydrophilic-lipophilic balance (HLB) number indicates the polarity ofthe molecule in an arbitrary range of 1-40, with the most commonly usedemulsifiers having a value between 1-20. The HLB increases withincreasing hydrophilicity.

Surfactants that may be used include but are not limited to those withan HLB greater than 10, 11, 12, 13 or 14. Examples of surfactantsinclude polyoxyethylene products of hydrogenated vegetable oils,polyethoxylated castor oils or polyethoxylated hydrogenated castor oil,polyoxyethylene-sorbitan-fatty acid esters, polyoxyethylene castor oilderivatives and the like, for example, Nikkol HCO-50, Nikkol HCO-35,Nikkol HCO-40, Nikkol HCO-60 (from Nikko Chemicals Co. Ltd.); Cremophor(from BASF) such as Cremophor RH40, Cremophor RH60, Cremophor EL, TWEENs(from ICI Chemicals) e.g., TWEEN 20, TWEEN 21, TWEEN 40, TWEEN 60, TWEEN80, TWEEN 81, Cremophor RH 410, Cremophor RH 455 and the like.

The surfactant component may be selected from compounds having at leastone ether formed from at least about 1 to 100 ethylene oxide units andat least one fatty alcohol chain having from at least about 12 to 22carbon atoms; compounds having at least one ester formed from at leastabout 1 to 100 ethylene oxide units and at least one fatty acid chainhaving from at least about 12 to 22 carbon atoms; compounds having atleast one ether, ester or amide formed from at least about 1 to 100ethylene oxide units and at least one vitamin or vitamin derivative; andcombinations thereof consisting of no more than two surfactants.

Other examples of surfactants include Lumulse GRH-40, TGPS,Polysorbate-80 (TWEEN-80), Polysorbate-20 (TWEEN-20), polyoxyethylene(20) sorbitan mono-oleate), glyceryl glycol esters, polyethylene glycolesters, polyglycolyzed glycerides, and the like, or mixtures thereof;polyethylene sorbitan fatty acid esters, polyoxyethylene glycerolesters, such as Tagat TO, Tagat L, Tagat I, tagat 12 and Tagat 0(commercially available from Goldschmidt Chemical Co., Essen, Germany);ethylene glycol esters, such as glycol stearate and distearate;propylene glycol esters, such as propylene glycol myristate; glycerylesters of fatty acids, such as glyceryl stearates and monostearates;sorbitan esters, such as spans and TWEENs; polyglyceryl esters, such aspolyglyceryl 4-oleate; fatty alcohol ethoxylates, such as Brij typeemulsifiers; ethoxylated propoxylated block copolymers, such aspoloxamers; polyethylene glycol esters of fatty acids, such as PEG 300μlinoleic glycerides or Labrafil 2125 CS, PEG 300 oleic glycerides orLabrafil M 1944 CS, PEG 400 caprylic/capric glycerides or Labrasol, andPEG 300 caprylic/capric glycerides or Softigen 767; cremophors, such asCremophor E, polyoxyl 35 castor oil or Cremophor EL, Cremophor EL-P,Cremophor RH 4OP, polyoxyl 40 hydrogenated castor oil, Cremophor RH40;polyoxyl 60 hydrogenated castor oil or Cremophor RH 60, glycerolmonocaprylate/caprate, such as Campmul CM 10; polyoxyethylated fattyacids (PEG-stearates, PED-laurates, Brij®), polyoxylated glycerides offatty acid, polyoxylated glycerol fatty acid esters i.e. Solutol HS-15;PEG-ethers (Mirj®), sorbitan derivatives (TWEENs), sorbitan monooleateor Span 20, aromatic compounds (Tritons®), PEG-glycerides (PECEOL™),PEG-PPG (polypropylene glycol) copolymers (PLURONICS including but notlimited to PLURONICS F108, F127, and F68, Poloxamers, Jeffamines),Tetronics, Polyglycerines, PEG-tocopherols, PEG-LICOL 6-oleate;propylene glycol derivatives, sugar and polysaccharide alkyl and acylderivatives (octylsucrose, sucrose stearate, laurolydextran etc.) and/ora mixture thereof; surfactants based on an oleate or laureate ester of apolyalcohol copolymerized with ethylene oxide; Labrasol Gelucire 44/14;polyoxytheylene stearates; saturated polyglycolyzed glycerides; orpoloxamers; all of which are commercially available. Polyoxyethylenesorbitan fatty acid esters can include polysorbates, for example,polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.Polyoxyethylene stearates can include polyoxyl 6 stearate, polyoxyl 8stearate, polyoxyl 12 stearate and polyoxyl 20 stearate. Saturatedpolyglycolyzed glycerides are, for example, GELUCIRE 44/14 or GELUCIRE™50/13 (Gattefosse, Westwood, N.J., U.S.A.). Poloxamers used hereininclude poloxamer 124 and poloxamer 188.

Surfactants include d-α-tocopheryl polyethylene glycol 1000 succinate(TPGS), polyoxyl 8 stearate (PEG 400 monostearate), polyoxyl 40 stearate(PEG 1750 monostearate) and peppermint oil.

In some variations, surfactants having an HLB lower than 10 are used.Such surfactants may optionally be used in combination with othersurfactants as co-surfactants. Examples of some surfactants, mixtures,and other equivalent compositions having an HLB less than or equal to 10are propylene glycols, glyceryl fatty acids, glyceryl fatty acid esters,polyethylene glycol esters, glyceryl glycol esters, polyglycolyzedglycerides and polyoxyethyl steryl ethers. Propylene glycol esters orpartial esters form the composition of commercial products, such asLauroglycol FCC, which contains propylene glycol laureate. Thecommercially available excipient Maisine 35-1 comprises long chain fattyacids, for example glyceryl linoleate. Products, such as Acconon E,which comprise polyoxyethylene stearyl ethers, may also be used.Labrafil M 1944 CS is one example of a surfactant wherein thecomposition contains a mixture of glyceryl glycol esters andpolyethylene glycol esters.

Solubilizing Agents for Rapamycin

Many solubilizing agents may be used for rapamycin, including but notlimited to those in the solubilizing agents section above.

In some variations the solubilizing agent is a surfactant. Nonlimitingexamples of surfactants that may be used for rapamycin include but arenot limited to surfactants with an HLB greater than 10, 11, 12, 13 or14. One nonlimiting example is Cremophor EL. In some variations, thesurfactant may be a polymeric surfactant including but not limited toPLURONICS F108, F127, and F68, and Tetronics. As noted herein, somesolvents may also serve as surfactants. Those of ordinary skill in theart will find it routine to identify which solubilizing agents andsurfactants may be used for rapamycin given the teachings herein.

Viscosity Modifying Agents

The liquid formulations described herein may be administered with orfurther comprise a viscosity modifying agent.

One exemplary viscosity modifying agent that may be used is hyaluronicacid. Hyaluronic acid is a glycosaminoglycan. It is made of a repetitivesequence of glucuronic acid and glucosamine. Hyaluronic acid is presentin many tissues and organs of the body, and contributes to the viscosityand consistency of such tissues and organs. Hyaluronic acid is presentin the eye, including the vitreous of the eye, and along with collagencontributes to the viscosity thereof. The liquid formulations describedherein may further comprise or be administered with hyaluronic acid.

Other nonlimiting examples of viscosity modifying agents includepolyalkylene oxides, glycerol, carboxymethyl cellulose, sodium alginate,chitosan, dextran, dextran sulfate and collagen. These viscositymodifying agents can be chemically modified.

Other viscosity modifying agents that may be used include but are notlimited to carrageenan, cellulose gel, colloidal silicon dioxide,gelatin, propylene carbonate, carbonic acid, alginic acid, agar,carboxyvinyl polymers or carbomers and polyacrylamides, acacia, estergum, guar gum, gum arabic, ghatti, gum karaya, tragacanth, terra,pectin, tamarind seed, larch arabinogalactan, alginates, locust bean,xanthan gum, starch, veegum, tragacanth, polyvinyl alcohol, gellan gum,hydrocolloid blends, and povidone. Other viscosity modifying agentsknown in the art can also be used, including but not limited to sodiumcarboxymethyl cellulose, algin, carageenans, galactomannans, hydropropylmethyl cellulose, hydroxypropyl cellulose, polyethylene glycol,polyvinylpyrrolidone, sodium carboxymethyl chitin, sodium carboxymethyldextran, sodium carboxymethyl starch, xanthan gum, and zein.

Other Components of Liquid Formulations

The formulations described herein may further comprise various othercomponents such as stabilizers, for example. Stabilizers that may beused in the formulations described herein include but are not limited toagents that will (1) improve the compatibility of excipients with theencapsulating materials such as gelatin, (2) improve the stability (e.g.prevent crystal growth of a therapeutic agent such as rapamycin) of atherapeutic agent such as rapamycin and/or rapamycin derivatives, and/or(3) improve formulation stability. Note that there is overlap betweencomponents that are stabilizers and those that are solvents,solubilizing agents or surfactants, and the same component can carry outmore than one role.

Stabilizers may be selected from fatty acids, fatty alcohols, alcohols,long chain fatty acid esters, long chain ethers, hydrophilic derivativesof fatty acids, polyvinylpyrrolidones, polyvinylethers, polyvinylalcohols, hydrocarbons, hydrophobic polymers, moisture-absorbingpolymers, and combinations thereof. Amide analogues of the abovestabilizers can also be used. The chosen stabilizer may change thehydrophobicity of the formulation (e.g. oleic acid, waxes), or improvethe mixing of various components in the formulation (e.g. ethanol),control the moisture level in the formula (e.g. PVP), control themobility of the phase (substances with melting points higher than roomtemperature such as long chain fatty acids, alcohols, esters, ethers,amides etc. or mixtures thereof; waxes), and/or improve thecompatibility of the formula with encapsulating materials (e.g. oleicacid or wax). Some of these stabilizers may be used assolvents/co-solvents (e.g. ethanol). Stabilizers may be present insufficient amount to inhibit the therapeutic agent's (such asrapamycin's) crystallization.

Examples of stabilizers include, but are not limited to, saturated,monoenoic, polyenoic, branched, ring-containing, acetylenic,dicarboxylic and functional-group-containing fatty acids such as oleicacid, caprylic acid, capric acid, caproic acid, lauric acid, myristicacid, palmitic acid, stearic acid, behenic acid, linoleic acid,linolenic acid, eicosapentaenoic acid (EPA), DHA; fatty alcohols such asstearyl alcohol, cetyl alcohol, ceteryl alcohol; other alcohols such asethanol, isopropyl alcohol, butanol; long chain fatty acid esters,ethers or amides such as glyceryl stearate, cetyl stearate, oleylethers, stearyl ethers, cetyl ethers, oleyl amides, stearyl amides;hydrophilic derivatives of fatty acids such as polyglyceryl fatty acids,polyethylene glycol fatty acid esters; polyvinylpyrrolidones,polyvinylalcohols (PVAs), waxes, docosahexaenoic acid andde-hydroabietic acid etc.

The formulations described may further contain a gelling agent thatalters the texture of the final formulation through formation of a gel.

The therapeutic agents for use as described herein, such as rapamycin,may be subjected to conventional pharmaceutical operations, such assterilization and compositions containing the therapeutic agent may alsocontain conventional adjuvants, such as preservatives, stabilizers,wetting agents, emulsifiers, buffers etc. The therapeutic agents mayalso be formulated with pharmaceutically acceptable excipients forclinical use to produce a pharmaceutical composition. Formulations forocular administration may be presented as a solution, suspension,particles of solid material, a discrete mass of solid material,incorporated within a polymer matrix, liquid formulations or in anyother form for ocular administration. The therapeutic agents may be usedto prepare a medicament to treat, prevent, inhibit, delay onset, orcause regression of any of the conditions described herein. In somevariations, the therapeutic agents may be used to prepare a medicamentto treat any of the conditions described herein.

A composition containing a therapeutic agent such as rapamycin maycontain one or more adjuvants appropriate for the indicated route ofadministration. Adjuvants with which the therapeutic agent may beadmixed with include but are not limited to lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulphuric acids, acacia, gelatin, sodium alginate,polyvinylpyrrolidine, and/or polyvinyl alcohol. When a solubilizedformulation is required the therapeutic agent may be in a solventincluding but not limited to polyethylene glycol of various molecularweights, propylene glycol, carboxymethyl cellulose colloidal solutions,methanol, ethanol, DMSO, corn oil, peanut oil, cottonseed oil, sesameoil, tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art and may beused in the practice of the methods, compositions and liquidformulations described herein. The carrier or diluent may include timedelay material, such as glyceryl monostearate or glyceryl distearatealone or with a wax, or other materials well known in the art. Theformulations for use as described herein may also include gelformulations, erodible and non-erodible polymers, microspheres, andliposomes.

Other adjuvants and excipients that may be used include but are notlimited to C₈-C₁₀ fatty acid esters such as softigen 767, polysorbate80, PLURONICS, Tetronics, Miglyol, and Transcutol.

Additives and diluents normally utilized in the pharmaceutical arts canoptionally be added to the pharmaceutical composition and the liquidformulation. These include thickening, granulating, dispersing,flavoring, sweetening, coloring, and stabilizing agents, including pHstabilizers, other excipients, anti-oxidants (e.g., tocopherol, BHA,BHT, TBHQ, tocopherol acetate, ascorbyl palmitate, ascorbic acid propylgallate, and the like), preservatives (e.g., parabens), and the like.Exemplary preservatives include, but are not limited to, benzylalcohol,ethylalcohol, benzalkonium chloride, phenol, chlorobutanol, and thelike. Some useful antioxidants provide oxygen or peroxide inhibitingagents for the formulation and include, but are not limited to,butylated hydroxytoluene, butylhydroxyanisole, propyl gallate, ascorbicacid palmitate, α-tocopherol, and the like. Thickening agents, such aslecithin, hydroxypropylcellulose, aluminum stearate, and the like, mayimprove the texture of the formulation.

In some variations, the therapeutic agent is rapamycin, and therapamycin is formulated as rapamune in solid or liquid form. In somevariations, the rapamune is formulated as an oral dosage.

In addition, a viscous polymer may be added to the suspension, assistingthe localization and ease of placement and handling. In some uses of theliquid formulation, a pocket in the sclera may be surgically formed toreceive an injection of the liquid formulations. The hydrogel structureof the sclera can act as a rate-controlling membrane. Particles oftherapeutic agent substance for forming a suspension can be produced byknown methods including but not limited to via ball milling, for exampleby using ceramic beads. For example, a Cole Parmer ball mill such asLabmill 8000 may be used with 0.8 mm YTZ ceramic beads available fromTosoh or Norstone Inc.

The formulations may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the therapeuticagent and the pharmaceutical carrier(s) or excipient(s). Theformulations may be prepared by uniformly and intimately bringing intoassociate the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

In some variations, the formulations described herein are provided inone or more unit dose forms, wherein the unit dose form contains anamount of a liquid formulation described herein that is effective totreat or prevent the disease or condition for which it is beingadministered. In some variations, the formulations described herein areprovided in one or more unit dose forms, wherein the unit dose formcontains an amount of a liquid rapamycin formulation described hereinthat is effective to treat or prevent the disease or condition for whichit is being administered.

In some embodiments, the unit dose form is prepared in the concentrationat which it will be administered. In some variations, the unit dose formis diluted prior to administration to a subject. In some variations, aliquid formulation described herein is diluted in an aqueous mediumprior to administration to a subject. In some variations the aqueousmedium is an isotonic medium. In some variations, a liquid formulationdescribed herein is diluted in an non-aqueous medium prior toadministration to a subject.

In a further aspect, provided herein are kits comprising one or moreunit dose forms as described herein. In some embodiments, the kitcomprises one or more of packaging and instructions for use to treat oneor more diseases or conditions. In some embodiments, the kit comprises adiluent which is not in physical contact with the formulation orpharmaceutical formulation. In some embodiments, the kit comprises anyof one or more unit dose forms described herein in one or more sealedvessels. In some embodiments, the kit comprises any of one or moresterile unit dose forms.

In some variations, the unit dose form is in a container, including butnot limited to a sterile sealed container. In some variations thecontainer is a vial, ampule, or low volume applicator, including but notlimited to a syringe. In some variations, a low-volume applicator ispre-filled with rapamycin for treatment of an ophthalmic disease orcondition, including but not limited to a limus compound for treatmentof age-related macular degeneration. Described herein is a pre-filledlow-volume applicator pre-filled with a formulation comprising atherapeutic agent, including but not limited to rapamycin. In somevariations a low-volume applicator is pre-filled with a solutioncomprising a therapeutic agent, including but not limited to rapamycinand a polyethylene glycol, and optionally further comprises one or moreadditional components including but not limited to ethanol. In somevariations a pre-filled low-volume applicator is pre-filled with asolution comprising about 2% rapamycin, about 94% PEG-400, about 4%ethanol.

Described herein are kits comprising one or more containers. In somevariations a kit comprises one or more low-volume applicators ispre-filled with a formulation described herein comprising a therapeuticagent, including but not limited to formulations comprising rapamycin,formulations comprising rapamycin and a polyethylene glycol, andoptionally further comprises one or more additional components includingbut not limited to ethanol, and formulations in liquid form comprisingabout 2% rapamycin, about 94% PEG-400, about 4% ethanol. In somevariations the kit comprises one or more containers, including but notlimited to pre-filled low-volume applicators, with instructions for itsuse. In a further variation a kit comprises one or more low-volumeapplicators pre-filled with rapamycin, with instructions for its use intreating a disease or condition of the eye. In some variations, thecontainers described herein are in a secondary packaging.

Routes of Administration

The compositions, methods, and liquid formulations described hereindeliver one or more therapeutic agents to a subject, including but notlimited to a human subject.

In some variations, the compositions, methods, and liquid formulationsdescribed herein deliver one or more therapeutic agents to an aqueousmedium of a human subject.

In some variations, the compositions, methods, and liquid formulationsdescribed herein deliver one or more therapeutic agents to an aqueousmedium in or proximal to an area where a disease or condition is to betreated, prevented, inhibited, onset delayed, or regression caused.

In some variations, the compositions, methods, and liquid formulationsdescribed herein deliver one or more therapeutic agents to an eye of asubject, including the macula and the retina choroid tissues, in anamount and for a duration effective to treat, prevent, inhibit, delaythe onset of, or cause the regression of the diseases and conditionsdescribed in the Diseases and Conditions section.

“Retina choroid” and “retina choroid tissues,” as used herein, aresynonymous and refer to the combined retina and choroid tissues of theeye.

As a non-limiting example, the compositions, liquid formulations, andmethods described in herein may be administered to the vitreous, aqueoushumor, sclera, conjunctiva, between the sclera and conjunctiva, theretina choroid tissues, macula, or other area in or proximate to the eyeof a subject, either by direct administration to these tissues or byperiocular routes, in amounts and for a duration effective to treat,prevent, inhibit, delay the onset of, or cause the regression of CNV andwet AMD. The effective amounts and durations may be different for eachof treating, preventing, inhibiting, delaying the onset of, or causingthe regression of CNV and wet AMD, and for each of the different sitesof delivery.

Intravitreal administration is more invasive than some other types ofocular procedures. Because of the potential risks of adverse effects,intravitreal administration may not be optimal for treatment ofrelatively healthy eyes. By contrast, periocular administration, such assubconjunctival administration, is much less invasive than intravitrealadministration. When a therapeutic agent is delivered by a periocularroute, it may be possible to treat patients with healthier eyes thancould be treated using intravitreal administration. In some variations,subconjunctival injection is used to prevent or delay onset of a diseaseor condition of the eye, where the eye of the subject has visual acuityof 20/40 or better.

“Subconjunctival” placement or injection, as used herein, refers toplacement or injection between the sclera and conjunctiva.Subconjunctival is sometimes referred to herein as “sub-conj”administration.

Routes of administration that may be used to administer a liquidformulation include but are not limited to placement of the liquidformulation, for example by injection, into an aqueous medium in thesubject, including but not limited to placement, including but notlimited to by injection, into the eye of a subject, including but notlimited to a human subject. The liquid formulation may be administeredsystemically, including but not limited to the following deliveryroutes: rectal, vaginal, infusion, intramuscular, intraperitoneal,intraarterial, intrathecal, intrabronchial, intracisternal, cutaneous,subcutaneous, intradermal, transdermal, intravenous, intracervical,intraabdominal, intracranial, intraocular, intrapulmonary,intrathoracic, intratracheal, nasal, buccal, sublingual, oral,parenteral, or nebulised or aerosolized using aerosol propellants.

Compositions and liquid formulations comprising therapeutic agent can beadministered directly to the eye using a variety of procedures,including but not limited to procedures in which (1) the therapeuticagent is administered by injection using a syringe and hypodermicneedle, (2) a specially designed device is used to inject thetherapeutic agent, (3) prior to injection of the therapeutic agent, apocket is surgically formed within the sclera to serve as a receptaclefor the therapeutic agent or therapeutic agent composition. For example,in one administration procedure a surgeon forms a pocket within thesclera of the eye followed by injection of a solution or liquidformulation comprising the therapeutic agent into the pocket.

Other administration procedures include, but are not limited toprocedures in which (1) a formulation of the therapeutic agent isinjected through a specially designed curved cannula to place thetherapeutic agent directly against a portion of the eye, (2) acompressed form of the therapeutic agent is placed directly against aportion of the eye, (3) the therapeutic agent is inserted into thesclera by a specially designed injector or inserter, (4) the liquidformulation comprising the therapeutic agent is incorporated within apolymer, (5) a surgeon makes a small conjunctival incision through whichto pass a suture and any therapeutic agent delivery structure so as tosecure the structure adjacent to the sclera, (6) a needle is used forinjection directly into the vitreous of an eye, or into any other sitedescribed.

The liquid formulations described herein may be used directly, forexample, by injection, as an elixir, for topical administrationincluding but not limited to via eye drops, or in hard or soft gelatinor starch capsules. The capsules may be banded to prevent leakage.

Delivery by Injection

One method that may be used to deliver the compositions and liquidformulations described herein is delivery by injection. In this methodcompositions and liquid formulations may be injected into a subject,including but not limited to a human subject, or into a position in orproximate to an eye of the subject for delivery to a subject or to theeye of a subject. Injection includes but is not limited to intraocularand periocular injection. Nonlimiting examples of positions that are inor proximate to an eye of a subject are as follows.

Injection of therapeutic agent into the vitreous may provide a highlocal concentration of therapeutic agent in the vitreous and retina.Further, it has been found that in the vitreous the clearance half-livesof drugs increases with molecular weight.

Intracameral injection, or injection into the anterior chamber of theyeye, may also be used. In one example, up to about 100 μl may beinjected intracamerally.

Periocular routes of delivery may deliver therapeutic agent to theretina without some of the risks of intravitreal delivery. Periocularroutes include but are not limited to subconjunctival, subtenon,retrobulbar, peribulbar and posterior juxtascleral delivery. A“periocular” route of administration means placement near or around theeye. For a description of exemplary periocular routes for retinal drugdelivery, see Periocular routes for retinal drug delivery, Raghava etal. (2004), Expert Opin. Drug Deliv. 1(1):99-114, which is incorporatedherein by reference in its entirety.

In some variations the liquid formulations described herein areadministered intraocularly. Intraocular administration includesplacement or injection within the eye, including in the vitreous.

Subconjunctival injection may be by injection of therapeutic agentunderneath the conjunctiva, or between the sclera and conjunctiva. Inone example, up to about 500 μl may be injected subconjunctivally. Asone nonlimiting example, a needle of up to about 25 to about 30 gaugeand about 30 mm long may be used. Local pressure to the subconjunctivalsite of therapeutic agent administration may elevate delivery of thetherapeutic agent to the posterior segment by reducing local choroidalblood flow.

Subtenon injection may be by injection of therapeutic agent into thetenon's capsule around the upper portion of the eye and into the “belly”of the superior rectus muscle. In one example, up to about 4 ml may beinjected subtenon. As one nonlimiting example, a blunt-tipped cannulaabout 2.5 cm long may be used.

Retrobulbar injection refers to injection into the conical compartmentof the four rectus muscles and their intermuscular septa, behind theglobe of the eye. In one example, up to about 5 ml may be injectedretrobulbarly. As one nonlimiting example, a blunt needle of about 25-or about 27-gauge may be used.

Peribulbar injection may be at a location external to the confines ofthe four rectus muscles and their intramuscular septa, i.e., outside ofthe muscle cone. A volume of, for example, up to about 10 ml may beinjected peribulbarly. As one nonlimiting example, a blunt-tippedcannula about 1.25 inches long and about 25-gauge may be used.

Posterior juxtascleral delivery refers to placement of a therapeuticagent near and above the macula, in direct contact with the outersurface of the sclera, and without puncturing the eyeball. In oneexample, up to about 500 ml may be injected posterior juxtasclerally. Asone nonlimiting example, a blunt-tipped curved cannula, speciallydesigned at 560, is used to place the therapeutic agent in an incisionin the sclera.

In some variations the liquid formulations described herein are injectedintraocularly. Intraocular injection includes injection within the eye.

Sites to which the compositions and liquid formulations may beadministered include but are not limited to the vitreous, aqueous humor,sclera, conjunctiva, between the sclera and conjunctiva, the retinachoroid tissues, macula, or other area in or proximate to the eye of asubject. Methods that may be used for placement of the compositions andliquid formulations include but are not limited to injection.

In one method that may be used, the therapeutic agent is dissolved in ansolvent or solvent mixture and then injected into or proximate to thevitreous, aqueous humor, sclera, conjunctiva, between the sclera andconjunctiva, the retina choroid tissues, macula, other area in orproximate to the eye of a subject, or other medium of a subject,according to any of the procedures mentioned above. In one such methodthat may be used, the therapeutic agent is rapamycin in a liquidformulation.

When the therapeutic agent is rapamycin, the compositions and liquidformulations may be used to deliver or maintain an amount of rapamycinin tissues of the eye, including without limitation retina, choroid, orthe vitreous, which amount is effective to treat AMD. In one nonlimitingexample, it is believed that a liquid formulation delivering rapamycinin an amount capable of providing a concentration of rapamycin of about0.1 pg/ml to about 2 μg/ml in the vitreous may be used for treatment ofwet AMD. In some nonlimiting examples, it is believed that a liquidformulation delivering a concentration of rapamycin of about 0.1 pg/mgto about 1 μg/mg in the retina choroid tissues may be used for treatmentof wet AMD. Other effective concentrations are readily ascertainable bythose of skill in the art based on the teachings described herein.

Method of Preparing Liquid Formulations

One nonlimiting method that may be used for preparing the liquidformulations described herein, including but not limited to liquidformulations comprising rapamycin, is by mixing a solvent and atherapeutic agent together at room temperature or at slightly elevatedtemperature until a solution or suspension is obtained, with optionaluse of a sonicator, and then cooling the formulation. Other componentsincluding but not limited to those described above may then be mixedwith the formulation. Other preparation methods that may be used aredescribed herein including in the examples, and those of skill in theart will be able to select other preparation methods based on theteachings herein.

Extended Delivery of Therapeutic Agents

Described herein are compositions and liquid formulations showing invivo delivery or clearance profiles with one or more of the followingcharacteristics. The delivery or clearance profiles are for clearance ofthe therapeutic agent in vivo after injection of the composition orliquid formulations subconjunctivally or into the vitreous of a rabbiteye. In some variations, the delivery or clearance profiles are forclearance of rapamycin in vivo after injection of the composition orliquid formulations subconjunctivally or into the vitreous of a rabbiteye. The volume of the rabbit vitreous is approximately 30-40% of thevolume of the human vitreous. The amount of therapeutic agent ismeasured using techniques as described in Example 2, but withoutlimitation to the formulation and therapeutic agent described in Example2.

In some variations, the therapeutic agents with the in vivo delivery orclearance profiles described herein include but are not limited to thosedescribed in the Therapeutic Agents section. In some variations thetherapeutic agent is rapamycin. In some variations, the liquidformulations described herein are used to deliver therapeutic agents ina concentration equivalent to rapamycin. The liquid formulationsdescribed herein may comprise any therapeutic agent including but notlimited to those in the Therapeutic Agents section, in a concentrationequivalent to rapamycin including but not limited to thoseconcentrations described herein including in the examples.

“Average percentage in vivo” level means that an average concentrationof therapeutic agent is obtained across multiple rabbit eyes for a giventimepoint, and the average concentration of therapeutic agent at onetimepoint is divided by the average concentration of therapeutic agentat another timepoint. In some variations of the average percentage invivo levels, the therapeutic agent is rapamycin.

The average concentration of a therapeutic agent in the tissue of arabbit eye at a given time after administration of a formulationcontaining the therapeutic agent may be measured according to thefollowing method. Where volumes below 10 μl are to be injected, aHamilton syringe is used.

The liquid formulations are stored at a temperature of 2-8° C. prior touse.

The experimental animals are specific pathogen free (SPF) New ZealandWhite rabbits. A mixed population of about 50% male, about 50% female isused. The rabbits are at least 12 weeks of age, usually at least 14weeks of age, at the time of dosing. The rabbits each weigh at least 2.2kg, usually at least 2.5 kg, at the time of dosing. Prior to the study,the animals are quarantined for at least one week and examined forgeneral health parameters. Any unhealthy animals are not used in thestudy. At least 6 eyes are measured and averaged for a given timepoint.

Housing and sanitation are performed according to standard proceduresused in the industry. The animals are provided approximately 150 gramsof Teklad Certified Hi-Fiber Rabbit Diet daily, and are provided tapwater ad libitum. No contaminants are known to exist in the water and noadditional analysis outside that provided by the local water district isperformed. Environmental Conditions are monitored.

Each animal undergoes a pre-treatment ophthalmic examination (slit lampand ophthalmoscopy), performed by a board certified veterinaryophthalmologist. Ocular findings are scored according to the McDonaldand Shadduck scoring system as described in Dermatoxicology, F. N.Marzulli and H. I. Maibach, 1977 “Eye Irritation,” T. O. McDonald and J.A. Shadduck (pages 579-582). Observations are recorded using astandardized data collection sheet. Acceptance criteria for placement onstudy are as follows: scores of <1 for conjunctival congestion andswelling; scores of 0 for all other observation variables.

Gentamicin ophthalmic drops are placed into both eyes of each animaltwice daily on the day prior to dosing, on the day of dosing (Day 1),and on the day after dosing (Day 2). Dosing is performed in two phases,the first including one set of animals and the second including theother animals. Animals are randomized separately into masked treatmentgroups prior to each phase of dosing according to modified Latinsquares. Animals are fasted at least 8 hours prior to injection. Thestart time of the fast and time of injection are recorded.

Animals are weighed and anesthetized with an intravenous injection of aketamine/xylazine cocktail (87 mg/mL ketamine, 13 mg/mL xylazine) at avolume of 0.1-0.2 mL/kg. Both eyes of each animal are prepared forinjection as follows: approximately five minutes prior to injection,eyes are moistened with an ophthalmic Betadine solution. After fiveminutes, the Betadine is washed out of the eyes with sterile saline.Proparacaine hydrochloride 0.5% (1-2 drops) is delivered to each eye.For eyes to be intravitreally injected, 1% Tropicamide (1 drop) isdelivered to each eye.

On Day 1, both eyes of each animal receive an injection of test orcontrol article. Animals in selected groups are dosed a second time onDay 90±1. Dosing is subconjunctival or intravitreal. Actual treatments,injection locations, and dose volumes are masked and revealed at the endof the study.

Subconjunctival injections are given using an insulin syringe and 30gauge×½-inch needle. The bulbar conjunctiva in the dorsotemporalquadrant is elevated using forceps. Test article is injected into thesubconjunctival space.

Intravitreal injections are given using an Insulin syringe and 30gauge×½-inch needle. For each injection, the needle is introducedthrough the ventral-nasal quadrant of the eye, approximately 2-3 mmposterior to the limbus, with the bevel of the needle directed downwardand posteriorly to avoid the lens. Test article is injected in a singlebolus in the vitreous near the retina.

Animals are observed for mortality/morbidity twice daily. An animaldetermined to be moribund is euthanized with an intravenous injection ofcommercial euthanasia solution. Both eyes are removed and stored frozenat −70° C. for possible future evaluation. If an animal is found deadprior to onset of rigor mortis, both eyes are removed and stored frozenat −70° C. for possible future evaluation. Animals found after the onsetof rigor mortis are not necropsied.

Animals are weighed at randomization, on Day 1 prior to dosing, andprior to euthanasia.

Ophthalmic observations (slit lamp and indirect ophthalmoscopy) areperformed on all animals on Days 5±1, 30±1, 60±1, 90±1, and at laterdates in some variations. Observations are performed by a boardcertified veterinary ophthalmologist. For animals to be dosed on Day90±1, ophthalmic observations are performed prior to dosing. Ocularfindings are scored according to the McDonald and Shadduck scoringsystem as described in Dermatoxicology, F. N. Marzulli and H. I.Maibach, 1977 “Eye Irritation”, T. O. McDonald and J. A. Shadduck (pages579-582), and observations are recorded using a standardized datacollection sheet.

Whole blood samples (1-3 mL per sample) are collected from each animalprior to necropsy in vacutainer tubes containing EDTA. Each tube isfilled at least ⅔ full and thoroughly mixed for at least 30 seconds.Tubes are stored frozen until shipped on dry ice.

Animals are euthanized with an intravenous injection of commercialeuthanasia solution. Euthanasia is performed according to standardprocedures used in the industry.

For treatment groups dosed intravitreally or subconjunctivally withplacebo, all eyes from each of these groups are placed into Davidsonssolution for approximately 24 hours. Following the 24-hour period, theeyes are transferred to 70% ethanol; these globes are submitted formasked histopathological evaluation by a board certified veterinarypathologist. The time that eyes are placed into Davidsons and the timeof removal are recorded.

For treatment groups dosed intravitreally or subconjunctivally with testarticle, some eyes from each of these groups are frozen at −70° C. andsubmitted for pharmacokinetic analysis. The remaining eyes from each ofthese groups are placed into Davidsons solution for approximately 24hours. Following the 24-hour period, the eyes are transferred to 70%ethanol; these globes are submitted for masked histopathologicalevaluation by a board certified veterinary pathologist. The time thateyes are placed into Davidsons and the time of removal are recorded.

Frozen samples submitted for pharmacokinetic analysis are dissected withdisposable instruments. One set of instruments is used per eye, and thendiscarded. The samples are thawed at room temperature for 1 to 2 minutesto ensure that the frost around the tissue has been removed. The sclerais dissected into 4 quadrants, and the vitreous is removed. If anon-dispersed mass (NDM) is clearly visible within the vitreous, thevitreous is separated into two sections. The section with the NDM isapproximately two-thirds of the vitreous. The section without the NDM isthe portion of the vitreous that is the most distant from the NDM. Theaqueous humor, lens, iris, and cornea are separated. The retina choroidtissue is removed using a forceps and collected for analysis. Theconjunctiva is separated from the sclera.

The various tissue types are collected into separate individualpre-weighed vials which are then capped and weighed. The vials of tissueare stored at −80° C. until analyzed.

The sirolimus content of the retina choroid, sclera, vitreous humor, andwhole anti-coagulated blood is determined by high-pressure liquidchromatography/tandem mass spectroscopy (HPLC/MS/MS) using32-O-desmethoxyrapamycin as an internal standard. Where an NDM wasobserved in the vitreous, the section of the vitreous containing the NDMand the section of the vitreous not containing the NDM are analyzedseparately.

The average concentration of a therapeutic agent over a period of timemeans for representative timepoints over the period of time the averageconcentration at each time point. For example, if the time period is 30days, the average concentration may be measured at 5 day intervals: forthe average concentration at day 5, the average of a number ofmeasurements of concentration at day 5 would be calculated; for theaverage concentration at day 10, the average of a number of measurementsof the concentration at day 10 would be calculated, etc.

In some variations, the liquid formulations described herein may have invivo delivery to the vitreous profiles with the following describedcharacteristics, where the delivery profiles are for delivery oftherapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye. One nonlimitingvariation of in vivo delivery to the vitreous profiles is shown in FIG.2.

At day 40 after injection, the average percentage in vivo vitreal levelmay be between about 70% and about 100%, and more usually between about80% and about 90%, relative to the level present at day 20 afterinjection. At day 40 after injection, the average percentage in vivovitreal level may be greater than about 70%, and more usually greaterthan about 80%, relative to the level present at day 20 after injection.

At day 67 after injection, the average percentage in vivo vitreal levelmay be between about 75% and about 115%, and more usually between about85% and about 105%, relative to the level present at day 20 afterinjection. At day 67 after injection, the average percentage in vivovitreal level may be greater than about 75%, and more usually greaterthan about 85%, relative to the level present at day 20 after injection.

At day 90 after injection, the average percentage in vivo vitreal levelmay be between about 20% and about 50%, and more usually between about30% and about 40%, relative to the level present at day 20 afterinjection. At day 90 after injection, the average percentage in vivovitreal level may be greater than about 20%, and more usually greaterthan about 30%, relative to the level present at day 20 after injection.

In some variations, the average percentage in vivo vitreal level has thefollowing characteristics relative to the level present at day 20 afterinjection: at 40 days after injection it is less than about 100%; at 67days after injection it is less than about 115%; and 90 days afterinjection it is less than about 50%.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least about 0.01 ng/mL for at least about 30, at leastabout 60, or at least about 90 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least about0.1 ng/mL for at least about 30, at least about 60, or at least about 90days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least about 1 ng/mL for at least about 30, at leastabout 60, or at least about 90 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulations described herein may have invivo delivery to the retina choroid profiles with the followingdescribed characteristics, where the delivery profiles are for deliveryof therapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye.

At day 40 after injection, the average percentage in vivo retina choroidlevel may be between about 350% and about 410%, and more usually betweenabout 360% and about 400%, relative to the level present at day 20 afterinjection. At day 40 after injection, the average percentage in vivoretina choroid level may be greater than about 350%, and more usuallygreater than about 360%, relative to the level present at day 20 afterinjection.

At day 67 after injection, the average percentage in vivo retina choroidlevel may be between about 125% and about 165%, and more usually betweenabout 135% and about 155%, relative to the level present at day 20 afterinjection. At day 67 after injection, the average percentage in vivoretina choroid level may be greater than about 125%, and more usuallygreater than about 135%, relative to the level present at day 20 afterinjection.

At day 90 after injection, the average percentage in vivo retina choroidlevel may be between about 10% and about 50%, and more usually betweenabout 20% and about 40%, relative to the level present at day 20 afterinjection. At day 90 after injection, the average percentage in vivoretina choroid level may be greater than about 10%, and more usuallygreater than about 20%, relative to the level present at day 20 afterinjection.

In some variations, the average percentage in vivo retina choroid levelhas the following characteristics relative to the level present at day20 after injection: at 40 days after injection it is less than about410%; at 67 days after injection it is less than about 165%; and 90 daysafter injection it is less than about 50%.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of at least about 0.001 ng/mg for at leastabout 30, at least about 60, or at least about 90 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of at least about 0.01 ng/mg for at least about 30, at leastabout 60, or at least about 90 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the level of therapeutic agent present in the retinachoroid first increases, then peaks and decreases. The peak may, forinstance, occur at about day 40 after injection.

In some variations, the liquid formulations described herein may have invivo clearance from the sclera profiles with the following describedcharacteristics, where the clearance profiles are for clearance oftherapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye. Where injectionis between the sclera and the conjunctiva, the scleral level is thoughtto include the injected liquid formulation.

At day 40 after injection, the average percentage in vivo scleral levelmay be between about 150% and about 230%, and more usually between about170% and about 210%, relative to the level present at day 20 afterinjection. At day 40 after injection, the average percentage in vivoscleral level may be greater than about 150%, and more usually greaterthan about 170%, relative to the level present at day 20 afterinjection.

At day 67 after injection, the average percentage in vivo scleral levelmay be between about 30% and about 70%, and more usually between about40% and about 60%, relative to the level present at day 20 afterinjection. At day 67 after injection, the average percentage in vivoscleral level may be greater than about 30%, and more usually greaterthan about 40%, relative to the level present at day 20 after injection.

At day 90 after injection, the average percentage in vivo scleral levelmay be between about 110% and about 160%, and more usually between about125% and about 145%, relative to the level present at day 20 afterinjection. At day 90 after injection, the average percentage in vivoscleral level may be greater than about 110%, and more usually greaterthan about 125%, relative to the level present at day 20 afterinjection.

In some variations, the average percentage in vivo scleral level has thefollowing characteristics relative to the level present at day 20 afterinjection: at 40 days after injection it is less than about 230%; at 67days after injection it is less than about 70%; and 90 days afterinjection it is less than about 160%.

In some variations, the level of therapeutic agent present in the sclerafirst increases, then peaks and decreases. The peak may, for instance,occur at about day 40 after injection.

In some variations, the liquid formulations described herein may have invivo delivery to the vitreous profiles with the following describedcharacteristics, where the delivery profiles are for delivery oftherapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye.

At day 14 after injection, the average percentage in vivo vitreal levelmay be between about 1350% and about 1650%, and more usually betweenabout 1450% and about 1550%, relative to the level present at day 2after injection. At day 14 after injection, the average percentage invivo vitreal level may be greater than about 1350%, and more usuallygreater than about 1450%, relative to the level present at day 2 afterinjection.

At day 35 after injection, the average percentage in vivo vitreal levelmay be between about 200% and about 300%, and more usually between about225% and about 275%, relative to the level present at day 2 afterinjection. At day 35 after injection, the average percentage in vivovitreal level may be greater than about 200%, and more usually greaterthan about 225%, relative to the level present at day 2 after injection.

At day 62 after injection, the average percentage in vivo vitreal levelmay be between about 100% and about 160%, and more usually between about115% and about 145%, relative to the level present at day 2 afterinjection. At day 62 after injection, the average percentage in vivovitreal level may be greater than about 100%, and more usually greaterthan about 115%, relative to the level present at day 2 after injection.

At day 85 after injection, the average percentage in vivo vitreal levelmay be between about 5% and about 30%, and more usually between about10% and about 25%, relative to the level present at day 2 afterinjection. At day 85 after injection, the average percentage in vivovitreal level may be greater than about 5%, and more usually greaterthan about 10%, relative to the level present at day 2 after injection.

In some variations, the average percentage in vivo vitreal level has thefollowing characteristics relative to the level present at day 2 afterinjection: at 14 days after injection it is less than about 1600%; at 35days after injection it is less than about 300%; at 62 days afterinjection it is less than about 160% and 85 days after injection it isless than about 30%.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least about 0.01 ng/mL for at least about 30, at leastabout 60, or at least about 85 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least about0.1 ng/mL for at least about 30, at least about 60, or at least about 85days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least about 1 ng/mL for at least about 30, or at leastabout 60 days after administration of the liquid formulation to therabbit eyes.

In some variations, the level of therapeutic agent present in thevitreous first increases, then peaks and decreases. The peak may, forinstance, occur at about day 14 after injection.

In some variations, the liquid formulations described herein may have invivo delivery to the retina choroid profiles with the followingdescribed characteristics, where the delivery profiles are for deliveryof therapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye.

At day 35 after injection, the average percentage in vivo retina choroidlevel may be between about 320% and about 400%, and more usually betweenabout 340% and about 380%, relative to the level present at day 14 afterinjection. At day 35 after injection, the average percentage in vivoretina choroid level may be greater than about 320%, and more usuallygreater than about 340%, relative to the level present at day 14 afterinjection.

At day 62 after injection, the average percentage in vivo retina choroidlevel may be between about 3% and about 25%, and more usually betweenabout 6% and about 20%, relative to the level present at day 14 afterinjection. At day 62 after injection, the average percentage in vivoretina choroid level may be greater than about 3%, and more usuallygreater than about 6%, relative to the level present at day 14 afterinjection.

At day 85 after injection, the average percentage in vivo retina choroidlevel may be between about 0.1% and about 6%, and more usually betweenabout 0.5% and about 4%, relative to the level present at day 14 afterinjection. At day 85 after injection, the average percentage in vivoretina choroid level may be greater than about 0.1%, and more usuallygreater than about 0.5%, relative to the level present at day 14 afterinjection.

In some variations, the average percentage in vivo retina choroid levelhas the following characteristics relative to the level present at day14 after injection: at 35 days after injection it is less than about400%; at 62 days after injection it is less than about 25%; and 85 daysafter injection it is less than about 6%.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of at least about 0.001 ng/mg for at leastabout 30, at least about 60, or at least about 85 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of at least about 0.01 ng/mg for at least about 30, at leastabout 60, or at least about 85 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulations described herein may have invivo clearance from the sclera profiles with the following describedcharacteristics, where the clearance profiles are for clearance oftherapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye. For injectionbetween the sclera and conjunctiva, the scleral level is thought toinclude the injected liquid formulation.

At day 35 after injection, the average percentage in vivo scleral levelmay be between about 0.1% and about 0.7%, and more usually between about0.2% and about 0.6%, relative to the level present at day 14 afterinjection. At day 35 after injection, the average percentage in vivoscleral level may be greater than about 0.1%, and more usually greaterthan about 0.2%, relative to the level present at day 14 afterinjection.

At day 62 after injection, the average percentage in vivo scleral levelmay be between about 0.05% and about 0.35%, and more usually betweenabout 0.07% and about 0.3%, relative to the level present at day 14after injection. At day 62 after injection, the average percentage invivo scleral level may be greater than about 0.05%, and more usuallygreater than about 0.07%, relative to the level present at day 14 afterinjection.

At day 85 after injection, the average percentage in vivo scleral levelmay be between about 0.1% and about 0.9%, and more usually between about0.3% and about 0.7%, relative to the level present at day 14 afterinjection. At day 85 after injection, the average percentage in vivoscleral level may be greater than about 0.1%, and more usually greaterthan about 0.3%, relative to the level present at day 14 afterinjection.

In some variations, the average percentage in vivo scleral level has thefollowing characteristics relative to the level present at day 14 afterinjection: at 35 days after injection it is less than about 0.7%; at 62days after injection it is less than about 0.35%; and 85 days afterinjection it is less than about 0.9%.

In some variations, the liquid formulations described herein may have invivo clearance from the vitreous profiles with the following describedcharacteristics, where the clearance profiles are for clearance oftherapeutic agent in vivo after injection of the liquid formulation intothe vitreous of a rabbit eye. Where injection is into the vitreous, themeasured vitreous level is thought to include the injected formulation.

At day 35 after injection, the average percentage in vivo vitreal levelmay be between about 1% and about 40%, and more usually between about 1%and about 10%, relative to the level present at day 14 after injection.At day 35 after injection, the average percentage in vivo vitreal levelmay be greater than about 1% relative to the level present at day 14after injection.

At day 62 after injection, the average percentage in vivo vitreal levelmay be between about 1% and about 40%, and more usually between about 5%and about 25%, relative to the level present at day 14 after injection.At day 62 after injection, the average percentage in vivo vitreal levelmay be greater than about 1% relative to the level present at day 14after injection, and more usually greater than about 5% relative to thelevel present at day 14 after injection.

At day 90 after injection, the average percentage in vivo vitreal levelmay be between about 1% and about 40%, and more usually between about10% and about 30%, relative to the level present at day 14 afterinjection. At day 90 after injection, the average percentage in vivovitreal level may be greater than about 1% relative to the level presentat day 14 after injection, and more usually greater than about 10%relative to the level present at day 14 after injection.

In some variations, the level of therapeutic agent present in thevitreous first increases, then peaks and decreases. The peak may, forinstance, occur at about day 14 after injection.

In some variations, the liquid formulations described herein may have invivo delivery to the retina choroid profiles with the followingdescribed characteristics, where the delivery profiles are for deliveryof therapeutic agent in vivo after injection of the liquid formulationinto the vitreous of a rabbit eye.

At day 35 after injection, the average percentage in vivo retina choroidlevel may be between about 3400% and about 5100%, and more usuallybetween about 3750% and about 4750%, relative to the level present atday 14 after injection. At day 35 after injection, the averagepercentage in vivo retina choroid level may be greater than about 3400%,and more usually greater than about 3750%, relative to the level presentat day 14 after injection.

At day 62 after injection, the average percentage in vivo retina choroidlevel may be between about 0.1% and about 5%, and more usually betweenabout 1% and about 3%, relative to the level present at day 14 afterinjection. At day 62 after injection, the average percentage in vivoretina choroid level may be greater than about 0.1%, and more usuallygreater than about 1%, relative to the level present at day 14 afterinjection.

At day 90 after injection, the average percentage in vivo retina choroidlevel may be between about 10% and about 50%, and more usually betweenabout 20% and about 40%, relative to the level present at day 14 afterinjection. At day 90 after injection, the average percentage in vivoretina choroid level may be greater than about 10%, and more usuallygreater than about 20%, relative to the level present at day 14 afterinjection.

In some variations, the average percentage in vivo retina choroid levelhas the following characteristics relative to the level present at day14 after injection: at 35 days after injection it is less than about5100%; at 62 days after injection it is less than about 5%; and 90 daysafter injection it is less than about 50%.

In some variations, the liquid formulations described herein may have invivo delivery to the sclera profiles with the following describedcharacteristics, where the delivery profiles are for delivery oftherapeutic agent in vivo after injection of the liquid formulation intothe vitreous of a rabbit eye.

At day 35 after injection, the average percentage in vivo scleral levelmay be between about 1700% and about 2600%, and more usually betweenabout 1900% and about 2400%, relative to the level present at day 14after injection. At day 35 after injection, the average percentage invivo scleral level may be greater than about 1700%, and more usuallygreater than about 1900%, relative to the level present at day 14 afterinjection.

At day 62 after injection, the average percentage in vivo scleral levelmay be between about 120% and about 180%, and more usually between about140% and about 160%, relative to the level present at day 14 afterinjection. At day 62 after injection, the average percentage in vivoscleral level may be greater than about 120%, and more usually greaterthan about 140%, relative to the level present at day 14 afterinjection.

At day 90 after injection, the average percentage in vivo scleral levelmay be between about 95% and about 155%, and more usually between about115% and about 135%, relative to the level present at day 14 afterinjection. At day 90 after injection, the average percentage in vivoscleral level may be greater than about 95%, and more usually greaterthan about 115%, relative to the level present at day 14 afterinjection.

In some variations, the average percentage in vivo scleral level has thefollowing characteristics relative to the level present at day 14 afterinjection: at 35 days after injection it is less than about 2600%; at 62days after injection it is less than about 180%; and 90 days afterinjection it is less than about 155%.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the sclera of the rabbit eye of atleast about 0.001 ng/mg for at least about 30, at least about 60, or atleast about 90 days after administration of the liquid formulation tothe rabbit eyes. In some variations, the liquid formulation wheninjected into the vitreous of a rabbit eye delivers therapeutic agentgiving an average concentration of therapeutic agent in the sclera ofthe rabbit eye of at least about 0.01 ng/mg for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the sclera of the rabbit eye of at least about 0.1 ng/mg for at leastabout 30, at least about 60, or at least about 90 days afteradministration of the liquid formulation to the rabbit eyes.

In some variations, the level of therapeutic agent present in thevitreous first increases, then peaks and decreases. The peak may, forinstance, occur at about day 35 after injection.

In some variations, in situ gelling liquid formulations described hereinmay have in vivo delivery to the vitreous profiles with the followingdescribed characteristics, where the delivery profiles are for deliveryof therapeutic agent in vivo after injection of the liquid formulationbetween the sclera and the conjunctiva of a rabbit eye.

At day 32 after injection, the average percentage in vivo vitreal levelmay be between about 25% and about 85%, and more usually between about45% and about 65%, relative to the level present at day 7 afterinjection. At day 40 after injection, the average percentage in vivovitreal level may be greater than about 25%, and more usually greaterthan about 45%, relative to the level present at day 7 after injection.

At day 45 after injection, the average percentage in vivo vitreal levelmay be between about 2% and about 50%, and more usually between about 8%and about 20%, relative to the level present at day 7 after injection.At day 67 after injection, the average percentage in vivo vitreal levelmay be greater than about 2%, and more usually greater than about 5%,relative to the level present at day 7 after injection.

At day 90 after injection, the average percentage in vivo vitreal levelmay be between about 40% and about 100%, and more usually between about60% and about 80%, relative to the level present at day 7 afterinjection. At day 90 after injection, the average percentage in vivovitreal level may be greater than about 40%, and more usually greaterthan about 60%, relative to the level present at day 7 after injection.

In some variations, the average percentage in vivo vitreal level has thefollowing characteristics relative to the level present at day 7 afterinjection: at 32 days after injection it is less than about 80%; at 45days after injection it is less than about 30%; and 90 days afterinjection it is less than about 100%.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least about 0.1 pg/mL for at least about 30, at leastabout 60, or at least about 90 days after administration of the liquidformulation to the rabbit eye. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least about0.01 ng/mL for at least about 30, at least about 60, or at least about90 days after administration of the liquid formulation to the rabbiteye. In some variations, the liquid formulation when injected betweenthe sclera and conjunctiva of a rabbit eye delivers therapeutic agentgiving an average concentration of therapeutic agent in the vitreous ofthe rabbit eye of at least about 0.1 ng/mL for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eye. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least about 1ng/mL for at least about 30, at least about 60, or at least about 90days after administration of the liquid formulation to the rabbit eye.In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least about 10 ng/mL for at least about 30, at leastabout 60, or at least about 90 days after administration of the liquidformulation to the rabbit eye.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of at least 0.001 ng/mL for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least 0.01ng/mL for at least 30, at least 60, at least 90, or at least 120 daysafter administration of the liquid formulation to the rabbit eyes. Insome variations, the liquid formulation when injected between the scleraand conjunctiva of a rabbit eye delivers therapeutic agent giving anaverage concentration of therapeutic agent in the vitreous of the rabbiteye of at least 0.1 ng/mL for at least 30, at least 60, at least 90, orat least 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving an average concentration of therapeutic agent in thevitreous of the rabbit eye of at least 0.5 ng/mL for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of between 0.001 ng/mL and 10.0 ng/mL for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of between 0.01ng/mL and 10 ng/mL for at least 30, at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving an average concentration of therapeutic agent in thevitreous of the rabbit eye of between 0.1 ng/mL and 10 ng/mL for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of therabbit eye of between 0.5 ng/mL and 10.0 ng/mL for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givinga ratio of a maximum average concentration of therapeutic agent in thevitreous of a rabbit eye to a minimum average concentration oftherapeutic agent in the vitreous of a rabbit eye less than 100 for days30 to at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving a ratio ofa maximum average concentration of therapeutic agent in the vitreous ofa rabbit eye to a minimum average concentration of therapeutic agent inthe vitreous of a rabbit eye less than 50 for days 30 to at least 60, atleast 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving a ratio of a maximum averageconcentration of therapeutic agent in the vitreous of a rabbit eye to aminimum average concentration of therapeutic agent in the vitreous of arabbit eye less than 10 for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving a ratio of a maximum average concentration of therapeuticagent in the vitreous of a rabbit eye to a minimum average concentrationof therapeutic agent in the vitreous of a rabbit eye less than 5 fordays 30 to at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes.

“Approximately constant,” as used herein, means that the average leveldoes not vary by more than one order of magnitude over the extendedperiod of time, i.e., the difference between the maximum and minimum isless than a 10-fold difference for measurements of the averageconcentration at times in the relevant period of time.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the vitreous of arabbit eye that is approximately constant at a value greater than 0.001ng/mL for days 30 to at least 60, at least 90, or at least 120 daysafter administration of the solution to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the vitreous of a rabbit eye thatis approximately constant at a value greater than 0.01 ng/mL for days 30to at least 60, at least 90, or at least 120 days after administrationof the liquid formulation to the rabbit eyes. In some variations, theliquid formulation when injected between the sclera and conjunctiva of arabbit eye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of a rabbit eye that is approximatelyconstant at a value greater than 0.1 ng/mL for days 30 to at least 60,at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of a rabbit eye that is approximatelyconstant at a value of 1.0 ng/mL for days 30 to at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of at least 0.001 ng/mg for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye of atleast 0.005 ng/mg for at least 30, at least 60, at least 90, or at least120 days after administration of the liquid formulation to the rabbiteyes. In some variations, the liquid formulation when injected betweenthe sclera and conjunctiva of a rabbit eye delivers therapeutic agentgiving an average concentration of therapeutic agent in the retinachoroid tissues of the rabbit eye of at least 0.01 ng/mg for at least30, at least 60, at least 90, or at least 120 days after administrationof the liquid formulation to the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of between 0.001 ng/mg and 1.0 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of between 0.001 ng/mg and 0.50 ng/mg for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye ofbetween 0.001 ng/mg and 0.15 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye ofbetween 0.001 ng/mg and 0.1 ng/mg for at least 30, at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of between 0.005 ng/mg and 1.0 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of between 0.005 ng/mg and 0.50 ng/mg for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye ofbetween 0.005 ng/mg and 0.15 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye ofbetween 0.005 ng/mg and 0.1 ng/mg for at least 30, at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of between 0.01 ng/mg and 1.0 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected between the sclera andconjunctiva of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of between 0.01 ng/mg and 0.50 ng/mg for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye ofbetween 0.01 ng/mg and 0.15 ng/mg for at least 30, at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes. In some variations, the liquid formulation wheninjected between the sclera and conjunctiva of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of the rabbit eye of between 0.01 ng/mgand 0.1 ng/mg for at least 30, at least 60, at least 90, or at least 120days after administration of the liquid formulation to the rabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givinga ratio of a maximum average concentration of therapeutic agent in theretina choroid tissues of a rabbit eye to a minimum averageconcentration of therapeutic agent in the retina choroid tissues of arabbit eye less than 100 for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving a ratio of a maximum average concentration of therapeuticagent in the retina choroid tissues of a rabbit eye to a minimum averageconcentration of therapeutic agent in the retina choroid tissues of arabbit eye less than 50 for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving a ratio of a maximum average concentration of therapeuticagent in the retina choroid tissues of a rabbit eye to a minimum averageconcentration of therapeutic agent in the retina choroid tissues of arabbit eye less than 10 for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving a ratio of a maximum average concentration of therapeuticagent in the retina choroid tissues of a rabbit eye to a minimum averageconcentration of therapeutic agent in the retina choroid tissues of arabbit eye less than 5 for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of a rabbit eye that is approximately constant at a valuegreater than 0.001 ng/mg for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers therapeuticagent giving an average concentration of therapeutic agent in the retinachoroid tissues of a rabbit eye that is approximately constant at avalue greater than 0.005 ng/mg for days 30 to at least 60, at least 90,or at least 120 days after administration of the liquid formulation tothe rabbit eyes. In some variations, the liquid formulation wheninjected between the sclera and conjunctiva of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of a rabbit eye that is approximatelyconstant at a value greater than 0.01 ng/mg for days 30 to at least 60,at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the vitreous of the rabbit eye ofat least 100 ng/mL for at least 30, at least 60, at least 90, or atleast 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedinto the vitreous of a rabbit eye delivers therapeutic agent giving anaverage concentration of therapeutic agent in the vitreous of the rabbiteye of at least 1000 ng/mL for at least 30, at least 60, at least 90, orat least 120 days after administration of the liquid formulation to therabbit eyes. In some variations, the liquid formulation when injectedinto the vitreous of a rabbit eye delivers therapeutic agent giving anaverage concentration of therapeutic agent in the vitreous of the rabbiteye of at least 10,000 ng/mL for at least 30, at least 60, at least 90,or at least 120 days after administration of the liquid formulation tothe rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the vitreous of the rabbit eyebetween 100 ng/mL and 100,000 ng/mL for day 30 to at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes. In some variations, the liquid formulation wheninjected into the vitreous of a rabbit eye delivers therapeutic agentgiving an average concentration of therapeutic agent in the vitreous ofthe rabbit eye between 100 ng/mL and 50,000 ng/mL for day 30 to at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the vitreous of the rabbit eyebetween 1000 ng/mL and 100,000 ng/mL for day 30 to at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes. In some variations, the liquid formulation wheninjected into the vitreous of a rabbit eye delivers therapeutic agentgiving an average concentration of therapeutic agent in the vitreous ofthe rabbit eye between 1000 ng/mL and 50,000 ng/mL for day 30 to atleast 60, at least 90, or at least 120 days after administration of theliquid formulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving a ratio of amaximum average concentration of therapeutic agent in the vitreous ofthe rabbit eye to a minimum average concentration of therapeutic agentin the vitreous of the rabbit eye less than 100 for days 30 to at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving a ratio of a maximum average concentration oftherapeutic agent in the vitreous of the rabbit eye to a minimum averageconcentration of therapeutic agent in the vitreous of the rabbit eyeless than 50 for days 30 to at least 60, at least 90, or at least 120days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving a ratio of amaximum average concentration of therapeutic agent in the vitreous ofthe rabbit eye to a minimum average concentration of therapeutic agentin the vitreous of the rabbit eye less than 10 for days 30 to at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the vitreous of the rabbit eyethat is approximately constant at a value greater than 100 ng/mL fordays 30 to at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected into the vitreous of arabbit eye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye that isapproximately constant at a value greater than 1000 ng/mL for days 30 toat least 60, at least 90, or at least 120 days after administration ofthe liquid formulation to the rabbit eyes. In some variations, theliquid formulation when injected into the vitreous of a rabbit eyedelivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye that isapproximately constant at a value greater than 10,000 ng/mL for days 30to at least 60, at least 90, or at least 120 days after administrationof the liquid formulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of at least 0.001 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of the rabbit eye of at least 0.01 ng/mgfor at least 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes. In somevariations, the liquid formulation when injected into the vitreous of arabbit eye delivers therapeutic agent giving an average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye of atleast 0.05 ng/mg for at least 30, at least 60, at least 90, or at least120 days after administration of the liquid formulation to the rabbiteyes. In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye of at least 0.10 ng/mg for at least 30, at least 60, at least90, or at least 120 days after administration of the liquid formulationto the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.001 ng/mg and 10.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of the rabbit eye between 0.001 ng/mg and5.00 ng/mg for at least 30, at least 60, at least 90, or at least 120days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.001 ng/mg and 1.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.01 ng/mg and 10.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of the rabbit eye between 0.01 ng/mg and5.00 ng/mg for at least 30, at least 60, at least 90, or at least 120days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.01 ng/mg and 1.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.05 ng/mg and 10.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of the rabbit eye between 0.05 ng/mg and5.00 ng/mg for at least 30, at least 60, at least 90, or at least 120days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.05 ng/mg and 1.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.10 ng/mg and 10.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye deliverstherapeutic agent giving an average concentration of therapeutic agentin the retina choroid tissues of the rabbit eye between 0.10 ng/mg and5.00 ng/mg for at least 30, at least 60, at least 90, or at least 120days after administration of the liquid formulation to the rabbit eyes.In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving an averageconcentration of therapeutic agent in the retina choroid tissues of therabbit eye between 0.10 ng/mg and 1.00 ng/mg for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidformulation to the rabbit eyes.

In some variations, the liquid formulation when injected into thevitreous of a rabbit eye delivers therapeutic agent giving a ratio of amaximum average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye to a minimum average concentration oftherapeutic agent in the retina choroid tissues of the rabbit eye lessthan 100 for days 30 to at least 60, at least 90, or at least 120 daysafter administration of the liquid formulation to the rabbit eyes. Insome variations, the liquid formulation when injected into the vitreousof a rabbit eye delivers therapeutic agent giving a ratio of a maximumaverage concentration of therapeutic agent in the retina choroid tissuesof the rabbit eye to a minimum average concentration of therapeuticagent in the retina choroid tissues of the rabbit eye less than 50 fordays 30 to at least 60, at least 90, or at least 120 days afteradministration of the liquid formulation to the rabbit eyes.

In some variations, in situ gelling liquid formulations described hereinmay have in vivo delivery to the retina choroid tissue profiles with thefollowing described characteristics, where the delivery profiles are fordelivery of therapeutic agent in vivo after injection of the liquidformulation between the sclera and the conjunctiva of a rabbit eye.

At day 32 after injection, the percentage in vivo vitreal level may bebetween about 20% and about 80%, and more usually between about 40% andabout 60%, relative to the level present at day 7 after injection. Atday 40 after injection, the percentage in vivo vitreal level may begreater than about 20%, and more usually greater than about 40%,relative to the level present at day 7 after injection.

At day 45 after injection, the percentage in vivo vitreal level may bebetween about 15% and about 55%, and more usually between about 25% andabout 45%, relative to the level present at day 7 after injection. Atday 67 after injection, the percentage in vivo vitreal level may begreater than about 15%, and more usually greater than about 25%,relative to the level present at day 7 after injection.

At day 90 after injection, the percentage in vivo vitreal level may bebetween about 60% and about 100%, and more usually between about 70% andabout 90%, relative to the level present at day 7 after injection. Atday 90 after injection, the percentage in vivo vitreal level may begreater than about 60%, and more usually greater than about 70%,relative to the level present at day 7 after injection.

In some variations, the percentage in vivo vitreal level has thefollowing characteristics relative to the level present at day 7 afterinjection: at 32 days after injection it is less than about 80%; at 45days after injection it is less than about 60%; and 90 days afterinjection it is less than about 100%.

In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers therapeutic agent givingan average concentration of therapeutic agent in the retina choroidtissues of the rabbit eye of at least about 0.1 pg/mg for at least about30, at least about 60, or at least about 90 days after administration ofthe liquid formulation to the rabbit eye. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least about0.01 ng/mg for at least about 30, at least about 60, or at least about90 days after administration of the liquid formulation to the rabbiteye. In some variations, the liquid formulation when injected betweenthe sclera and conjunctiva of a rabbit eye delivers therapeutic agentgiving an average concentration of therapeutic agent in the vitreous ofthe rabbit eye of at least about 0.1 ng/mg for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eye. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers therapeutic agent giving an average concentration oftherapeutic agent in the vitreous of the rabbit eye of at least about 1ng/mL for at least about 30, at least about 60, or at least about 90days after administration of the liquid formulation to the rabbit eye.

In some variations, the ratio of the base ten logarithms of the averagelevels of a therapeutic agent in two or more of the retina choroidtissues, the sclera, and the vitreous is approximately constant over anextended period of time after placement of the in situ gellingformulation in or proximate to the eye. In some variations, the ratio ofthe base ten logarithms of the average levels of a therapeutic agent intwo or more of the retina choroid tissues, the sclera, and the vitreousis approximately constant over an extended period of time afterplacement of the in situ gelling formulation between the sclera and theconjunctiva of an eye. In some variations, the ratio of the base tenlogarithms of the average levels of a therapeutic agent in the vitreousand the sclera is approximately constant over an extended period of timeafter placement of the in situ gelling formulation between the scleraand the conjunctiva of an eye.

In some variations, the ratio of the base ten logarithms of the averagelevels of a therapeutic agent in the vitreous and the retina choroidtissues is approximately constant over an extended period of time. Putanother way, as the level of therapeutic agent in the vitreous rises,the level of therapeutic agent in the retina choroid tissues rises to asimilar degree when considered on the logarithmic scale, and vice versa.

In some variations, the ratio of the base ten logarithms of the averagelevels of a therapeutic agent in the vitreous versus the retina choroidtissues is approximately constant over an extended period of time ofabout 7, about 30, about 60, or about 90 days. In some variations, theratio of the average level of therapeutic agent in the vitreous relativeto the level of therapeutic agent in the retina choroid tissues afterplacement of the in situ gelling formulation between the sclera and theconjunctiva of an eye is constant at about 37:1 at day 7, about 40:1 atday 32, about 10:1 at day 45, and about 34:1 at day 90.

In some variations, the ratio of the average level of therapeutic agentin the vitreous relative to the level of therapeutic agent in the retinachoroid tissues is constant at about 40:1 over a period of about 7,about 32, about 45, or about 90 days.

In some variations, the average level of the therapeutic agent in any orall of the retina choroid tissues, the sclera, and the vitreous isapproximately constant over an extended period of time after placementof the in situ gelling formulation in or proximate to the eye.

In some variations, after placement of an in situ gelling formulationbetween the sclera and the conjunctiva, the average level of therapeuticagent in the vitreous is approximately constant at about 8.1 ng/ml. Insome variations, after placement of an in situ gelling formulationbetween the sclera and the conjunctiva, the average level of therapeuticagent in the retina choroid tissues is approximately constant at about0.25 ng/mg. In some variations, after placement of an in situ gellingformulation between the sclera and the conjunctiva, the average level oftherapeutic agent in the sclera is approximately constant at about 1930ng/mg.

In some variations, the in situ gelling formulation when injectedbetween the sclera and conjunctiva of a rabbit eye maintains an averagelevel of therapeutic agent in the vitreous that is approximatelyconstant at about 0.1 pg/mL for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the vitreous that is approximatelyconstant at about 0.001 ng/mL for at least about 30, at least about 60,or at least about 90 days after administration of the liquid formulationto the rabbit eye. In some variations, the in situ gelling formulationwhen injected between the sclera and conjunctiva of a rabbit eyemaintains an average level of therapeutic agent in the vitreous that isapproximately constant at about 0.01 ng/mL for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eye. In some variations, the in situgelling formulation when injected between the sclera and conjunctiva ofa rabbit eye maintains an average level of therapeutic agent in thevitreous that is approximately constant at about 0.1 ng/mL for at leastabout 30, at least about 60, or at least about 90 days afteradministration of the liquid formulation to the rabbit eye. In somevariations, the in situ gelling formulation when injected between thesclera and conjunctiva of a rabbit eye maintains an average level oftherapeutic agent in the vitreous that is approximately constant atabout 1 ng/mL for at least about 30, at least about 60, or at leastabout 90 days after administration of the liquid formulation to therabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the vitreous that is approximatelyconstant at about 10 ng/mL for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the vitreous that is approximatelyconstant at about 100 ng/mL for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye.

In some variations, the in situ gelling formulation when injectedbetween the sclera and conjunctiva of a rabbit eye maintains an averagelevel of therapeutic agent in the retina choroid tissues that isapproximately constant at about 0.1 pg/mg for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eye. In some variations, the in situgelling formulation when injected between the sclera and conjunctiva ofa rabbit eye maintains an average level of therapeutic agent in theretina choroid tissues that is approximately constant at about 0.001ng/mg for at least about 30, at least about 60, or at least about 90days after administration of the liquid formulation to the rabbit eye.In some variations, the in situ gelling formulation when injectedbetween the sclera and conjunctiva of a rabbit eye maintains an averagelevel of therapeutic agent in the retina choroid tissues that isapproximately constant at about 0.01 ng/mg for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eye. In some variations, the in situgelling formulation when injected between the sclera and conjunctiva ofa rabbit eye maintains an average level of therapeutic agent in theretina choroid tissues that is approximately constant at about 0.1 ng/mgfor at least about 30, at least about 60, or at least about 90 daysafter administration of the liquid formulation to the rabbit eye. Insome variations, the in situ gelling formulation when injected betweenthe sclera and conjunctiva of a rabbit eye maintains an average level oftherapeutic agent in the retina choroid tissues that is approximatelyconstant at about 1 ng/mg for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the retina choroid tissues that isapproximately constant at about 10 ng/mg for at least about 30, at leastabout 60, or at least about 90 days after administration of the liquidformulation to the rabbit eye.

In some variations, the in situ gelling formulation when injectedbetween the sclera and conjunctiva of a rabbit eye maintains an averagelevel of therapeutic agent in the sclera that is approximately constantat about 0.1 pg/mg for at least about 30, at least about 60, or at leastabout 90 days after administration of the liquid formulation to therabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the sclera that is approximatelyconstant at about 0.001 ng/mg for at least about 30, at least about 60,or at least about 90 days after administration of the liquid formulationto the rabbit eye. In some variations, the in situ gelling formulationwhen injected between the sclera and conjunctiva of a rabbit eyemaintains an average level of therapeutic agent in the sclera that isapproximately constant at about 0.01 ng/mg for at least about 30, atleast about 60, or at least about 90 days after administration of theliquid formulation to the rabbit eye. In some variations, the in situgelling formulation when injected between the sclera and conjunctiva ofa rabbit eye maintains an average level of therapeutic agent in thesclera that is approximately constant at about 0.1 ng/mg for at leastabout 30, at least about 60, or at least about 90 days afteradministration of the liquid formulation to the rabbit eye. In somevariations, the in situ gelling formulation when injected between thesclera and conjunctiva of a rabbit eye maintains an average level oftherapeutic agent in the sclera that is approximately constant at about1 ng/mg for at least about 30, at least about 60, or at least about 90days after administration of the liquid formulation to the rabbit eye.In some variations, the in situ gelling formulation when injectedbetween the sclera and conjunctiva of a rabbit eye maintains an averagelevel of therapeutic agent in the sclera that is approximately constantat about 10 ng/mg for at least about 30, at least about 60, or at leastabout 90 days after administration of the liquid formulation to therabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the sclera that is approximatelyconstant at about 100 ng/mg for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the sclera that is approximatelyconstant at about 1 ag/mg for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye. In some variations, the in situ gelling formulation wheninjected between the sclera and conjunctiva of a rabbit eye maintains anaverage level of therapeutic agent in the sclera that is approximatelyconstant at about 10 μg/mg for at least about 30, at least about 60, orat least about 90 days after administration of the liquid formulation tothe rabbit eye.

For treatment, prevention, inhibition, delaying the onset of, or causingthe regression of certain diseases or conditions, it may be desirable tomaintain delivery of a therapeutically effective amount of thetherapeutic agent for an extended period of time. Depending on thedisease or condition being treated, prevented, inhibited, having onsetdelayed, or being caused to regress this extended period of time may beat least about 1 week, at least about 2 weeks, at least about 3 weeks,at least about 1 month, at least about 3 months, at least about 6months, at least about 9 months, or at least about 1 year. Generally,however, any extended period of delivery may be possible. Atherapeutically effective amount of agent may be delivered for anextended period by a liquid formulation or composition that maintainsfor the extended period a concentration of agent in a subject or an eyeof a subject sufficient to deliver a therapeutically effective amount ofagent for the extended time.

Delivery of a therapeutically effective amount of the therapeutic agentfor an extended period may be achieved via placement of one compositionor liquid formulation or may be achieved by application of two or moredoses of composition or liquid formulations. As a non-limiting exampleof such multiple applications, maintenance of the therapeutic amount ofrapamycin for 3 months for treatment, prevention, inhibition, delay ofonset, or cause of regression of wet AMD may be achieved by applicationof one liquid formulation or composition delivering a therapeutic amountfor 3 months or by sequential application of a plurality of liquidformulations or compositions. The optimal dosage regime will depend onthe therapeutic amount of the therapeutic agent needing to be delivered,and the period over which it need be delivered. Those versed in suchextended therapeutic agent delivery dosing will understand how toidentify dosing regimes that may be used based on the teachings providedherein.

When using certain therapeutic agents or for the treatment, prevention,inhibition, delaying the onset of, or causing the regression of certaindiseases, it may be desirable for delivery of the therapeutic agent notto commence immediately upon placement of the liquid formulation orcomposition into the eye region, but for delivery to commence after somedelay. For example, but in no way limiting, such delayed release may beuseful where the therapeutic agent inhibits or delays wound healing anddelayed release is desirable to allow healing of any wounds occurringupon placement of the liquid formulation or composition. Depending onthe therapeutic agent being delivered and/or the diseases and conditionsbeing treated, prevented, inhibited, onset delayed, and regressioncaused this period of delay before delivery of the therapeutic agentcommences may be about 1 hour, about 6 hours, about 12 hours, about 18hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 8 days, about 9 days, about 10days, about 11 days, about 12 days, about 13 days, about 14 days, about21 days, about 28 days, about 35 days, or about 42 days. Other delayperiods may be possible. Delayed release formulations that may be usedare known to people versed in the technology.

Intravitreal and Subconjunctival Delivery of Rapamycin for Treatment,Prevention, Inhibition, Delay of Onset, or Cause of Regression of AMD

In one method described herein, a liquid formulation comprisingrapamycin is delivered subconjunctivally or to the vitreous of an eye toprevent, treat, inhibit, delay onset of, or cause regression ofangiogenesis in the eye, including but not limited to treating CNV asobserved, for example, in AMD. In some variations, the liquidformulation is used to treat angiogenesis in the eye, including but notlimited to treating CNV as observed, for example, in AMD. Rapamycin hasbeen shown to inhibit CNV in rat and mice models, as described in U.S.application Ser. No. 10/665,203, which is incorporated herein byreference in its entirety. Rapamycin has been observed to inhibitMatrigel™ and laser-induced CNV when administered systemically andsubretinally. Also, periocular injection of rapamycin inhibitslaser-induced CNV.

Other therapeutic agents that may be delivered to the eye, particularlythe vitreous of an eye, for treatment, prevention, inhibition, delayingonset, or causing regression of angiogenesis in the eye (such as CNV)are members of the limus family of compounds other than rapamycinincluding but not limited to everolimus and tacrolimus (FK-506).

As described herein, the dosage of the therapeutic agent will depend onthe condition being addressed, whether the condition is to be treated,prevented, inhibited, have onset delayed, or be caused to regress, theparticular therapeutic agent, and other clinical factors such as weightand condition of the subject and the route of administration of thetherapeutic agent. It is to be understood that the methods, liquidformulations, and compositions described herein have application forboth human and veterinary use, as well as uses in other possibleanimals. As described herein, tissue concentrations of therapeuticagents expressed in units of mass per volume generally refer to tissuesthat are primarily aqueous such as the vitreous, for example. Tissueconcentrations of therapeutic agents expressed in unit of mass per massgenerally refer to other tissues such as the sclera or retina choroidtissues, for example.

One concentration of rapamycin that may be used in the methods describedherein is one that provides about 0.01 pg/ml or pg/mg or more ofrapamycin at the tissue level. Another concentration that may be used isone that provides about 0.1 pg/ml or ng/mg or more at the tissue level.Another concentration that may be used is one that provides about 1pg/ml or ng/mg or more at the tissue level. Another concentration thatmay be used is one that provides about 0.01 ng/ml or ng/mg or more atthe tissue level. Another concentration that may be used is one thatprovides about 0.1 ng/ml or ng/mg or more at the tissue level. Anotherconcentration that may be used is one that provides about 0.5 ng/ml orng/mg or more at the tissue level. Another concentration that may beused is one that provides about 1 ng/ml or more at the tissue level.Another concentration that may be used is one that provides about 2ng/ml or more at the tissue level. Another concentration that may beused is one that provides about 3 ng/ml or more at the tissue level.Another concentration that may be used is one that provides about 5ng/ml or more at the tissue level. Another concentration that may beused is one that provides about 10 ng/ml or more at the tissue level.Another concentration that may be used is one that provides about 15ng/ml or more at the tissue level. Another concentration that may beused is one that provides about 20 ng/ml or more at the tissue level.Another concentration that may be used is one that provides about 30ng/ml or more at the tissue level. Another concentration that may beused is one that provides about 50 ng/ml or more at the tissue level.One of ordinary skill in the art would know how to arrive at anappropriate concentration depending on the route and duration ofadministration utilized, given the teachings herein.

Generally, the amount of rapamycin administered in a liquid formulationis an amount sufficient to treat, prevent, inhibit, delay the onset, orcause regression of the disease or condition of the eye for the requiredamount of time. In some variations the amount of rapamycin administeredin the liquid formulation is an amount sufficient to treat the diseaseor condition of the eye for the required amount of time.

In some variations, a total amount of rapamycin less than about 5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 5.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 4.5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 4.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 3.5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 3.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 2.5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 2 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 1.2 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 1.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 0.8 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 0.6 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 0.4 mg isadministered subconjunctivally. In some variations, a volume of aformulation is administered that contains an amount of rapamycindescribed herein.

In some variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 0.5% and about 6% issubconjunctivally administered to a human subject by administeringbetween about 0.1 μl and about 200 μl of a liquid formulation describedherein. In some variations, a liquid formulation containing aconcentration of rapamycin by weight of the total of between about 0.5%and about 4% is subconjunctivally administered to a human subject byadministering between about 1 μl and about 50 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing aconcentration of rapamycin by weight of the total of between about 1.5%and about 3.5% is subconjunctivally administered to a human subject byadministering between about 1 μl and about 15 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing aconcentration of rapamycin by weight of the total of about 2% issubconjunctivally administered to a human subject by administeringbetween about 1 μl and about 15 μl of a liquid formulation describedherein.

In some variations, a liquid formulation containing an amount ofrapamycin of between about 0.2 μg and about 4 mg is subconjunctivallyadministered to a human subject by administering between about 0.1 μland about 200 μl of a liquid formulation described herein. In somevariations, a liquid formulation containing an amount of rapamycin ofbetween about 20 μg and about 2 mg is subconjunctivally administered toa human subject by administering between about 1 μl and about 100 μl ofa liquid formulation described herein. In some variations, a liquidformulation containing an amount of rapamycin of between about 20 μg andabout 1 mg is subconjunctivally administered to a human subject byadministering between about 1 μl and about 50 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing anamount of rapamycin of between about 20 μg and about 500 μg issubconjunctivally administered to a human subject by administeringbetween about 1 μl and about 25 μl of a liquid formulation describedherein. In some variations, a liquid formulation containing an amount ofrapamycin of between about 20 μg and about 300 μg is subconjunctivallyadministered to a human subject by administering between about 1 μl andabout 15 μl of a liquid formulation described herein.

In some variations, a total amount of rapamycin less than about 200 μgis administered intravitreally. In some variations, a total amount ofrapamycin less than about 200 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 300 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 400 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 500 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 600 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 800 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 1 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 2 mg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 2.5 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 3 mg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 3.5 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 4 mg isadministered intravitreally. In some variations, a volume of aformulation is administered that contains an amount of rapamycindescribed herein.

In some variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 0.5% and about 6% isintravitreally administered to a human subject by administering betweenabout 0.1 μl and about 200 μl of a liquid formulation described herein.In some variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 0.5% and about 4% isintravitreally administered to a human subject by administering betweenabout 1 μl and about 50 μl of a liquid formulation described herein. Insome variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 1.5% and about 3.5% isintravitreally administered to a human subject by administering betweenabout 1 μl and about 15 μl of a liquid formulation described herein. Insome variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of about 2% is intravitreallyadministered to a human subject by administering between about 1 μl andabout 15 μl of a liquid formulation described herein.

In some variations, a liquid formulation containing an amount ofrapamycin of between about 0.2 μg and about 4 mg is intravitreallyadministered to a human subject by administering between about 0.1 μland about 200 μl of a liquid formulation described herein. In somevariations, a liquid formulation containing an amount of rapamycin ofbetween about 20 μg and about 2 mg is intravitreally administered to ahuman subject by administering between about 1 μl and about 100 μl of aliquid formulation described herein. In some variations, a liquidformulation containing an amount of rapamycin of between about 20 μg andabout 1 mg is intravitreally administered to a human subject byadministering between about 1 μl and about 50 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing anamount of rapamycin of between about 20 μg and about 500 μg isintravitreally administered to a human subject by administering betweenabout 1 μl and about 25 μl of a liquid formulation described herein. Insome variations, a liquid formulation containing an amount of rapamycinof between about 20 μg and about 300 μg is intravitreally administeredto a human subject by administering between about 1 μl and about 15 μlof a liquid formulation described herein.

In some variations a liquid formulation as described herein containingan amount of rapamycin of between about 1 μg and about 5 mg isadministered to a human subject for treatment of wet AMD. In somevariations a liquid formulation as described herein containing an amountof rapamycin of between about 20 μg and about 4 mg is administered to ahuman subject for treatment of wet AMD. In some variations a liquidformulation as described herein containing an amount of rapamycin ofbetween about 20 μg and about 1.2 mg is administered to a human subjectfor treatment of wet AMD. In some variations an amount of rapamycin ofbetween about 10 μg and about 0.5 mg is administered to a human subjectfor treatment of wet AMD. In some variations an amount of rapamycin ofbetween about 10 μg and 90 μg is administered to a human subject fortreatment of wet AMD. In some variations an amount of rapamycin ofbetween about 60 μg and about 120 μg is administered to a human subjectfor treatment of wet AMD. In some variations an amount of rapamycin ofbetween about 100 μg and about 400 μg is administered to a human subjectfor treatment of wet AMD. In some variations an amount of rapamycin ofbetween about 400 μg and about 1 mg is administered to a human subjectfor treatment of wet AMD. In some variations an amount of rapamycin ofbetween about 1 mg and about 5 mg is administered to a human subject fortreatment of wet AMD. In some variations, an amount of rapamycin ofbetween about 3 mg and about 7 mg is administered to a human subject fortreatment of wet AMD. In some variations, an amount of rapamycin ofbetween about 5 mg and about 10 mg is administered to a human subjectfor treatment of wet AMD.

In some variations a liquid formulation as described herein containingan amount of rapamycin of between about 1 μg and about 5 mg isadministered to a human subject for prevention of wet AMD. In somevariations a liquid formulation as described herein containing an amountof rapamycin of between about 20 μg and about 4 mg is administered to ahuman subject for prevention of wet AMD. In some variations a liquidformulation as described herein containing an amount of rapamycin ofbetween about 20 μg and about 1.2 mg is administered to a human subjectfor prevention of wet AMD. In some variations an amount of rapamycin ofbetween about 10 pg and about 0.5 mg is administered to a human subjectfor prevention of wet AMD. In some variations an amount of rapamycin ofbetween about 10 μg and 90 μg is administered to a human subject forprevention of wet AMD. In some variations an amount of rapamycin ofbetween about 60 μg and about 120 μg is administered to a human subjectfor prevention of wet AMD. In some variations an amount of rapamycin ofbetween about 100 μg and about 400 μg is administered to a human subjectfor prevention of wet AMD. In some variations an amount of rapamycin ofbetween about 400 μg and about 1 mg is administered to a human subjectfor prevention of wet AMD. In some variations an amount of rapamycin ofbetween about 1 mg and about 5 mg is administered to a human subject forprevention of wet AMD. In some variations, an amount of rapamycin ofbetween about 3 mg and about 7 mg is administered to a human subject forprevention of wet AMD. In some variations, an amount of rapamycin ofbetween about 5 mg and about 10 mg is administered to a human subjectfor prevention of wet AMD.

In some variations a liquid formulation as described herein containingan amount of rapamycin of between about 1 μg and about 5 mg isadministered to a human subject for treatment of dry AMD. In somevariations a liquid formulation as described herein containing an amountof rapamycin of between about 20 μg and about 4 mg is administered to ahuman subject for treatment of dry AMD. In some variations a liquidformulation as described herein containing an amount of rapamycin ofbetween about 20 μg and about 1.2 mg is administered to a human subjectfor treatment of dry AMD. In some variations an amount of rapamycin ofbetween about 10 μg and about 0.5 mg is administered to a human subjectfor treatment of dry AMD. In some variations an amount of rapamycin ofbetween about 10 μg and 90 μg is administered to a human subject fortreatment of dry AMD. In some variations an amount of rapamycin ofbetween about 60 μg and about 120 μg is administered to a human subjectfor treatment of dry AMD. In some variations an amount of rapamycin ofbetween about 100 μg and about 400 μg is administered to a human subjectfor treatment of dry AMD. In some variations an amount of rapamycin ofbetween about 400 μg and about 1 mg is administered to a human subjectfor treatment of dry AMD. In some variations an amount of rapamycin ofbetween about 1 mg and about 5 mg is administered to a human subject fortreatment of dry AMD. In some variations, an amount of rapamycin ofbetween about 3 mg and about 7 mg is administered to a human subject fortreatment of dry AMD. In some variations, an amount of rapamycin ofbetween about 5 mg and about 10 mg is administered to a human subjectfor treatment of dry AMD.

In some variations, a liquid formulation as described herein containingan amount of rapamycin of between about 1 μg and about 5 mg isadministered to a human subject for treatment of angiogenesis, includingbut not limited to choroidal neovascularization. In some variations, anamount of rapamycin of between about 20 μg and about 4 mg isadministered to the human subject; between about 20 μg and about 1.2 mg;between about 10 μg and about 0.5 mg is administered to a human subjectfor treatment of wet AMD, between about 10 μg and 90 μg, between about60 μg and 120 μg is administered to the human subject; between about 100μg and 400 μg, between about 400 μg and 1 mg is administered to thehuman subject; in some variations, an amount of rapamycin of betweenabout 1 mg and 5 mg is administered to the human subject; in somevariations, an amount of rapamycin of between about 3 mg and 7 mg isadministered to the human subject; in some variations, an amount ofrapamycin of between about 5 mg and 10 mg is administered to the humansubject for treatment of angiogenesis, including but not limited tochoroidal neovascularization.

In one method, a liquid formulation as described herein contains anamount of a therapeutic agent equivalent to an amount of rapamycin.

In one method, a liquid formulation as described herein containing anamount of a therapeutic agent equivalent to an amount of rapamycin ofbetween about 1 μg and about 5 mg is administered to a human subject fortreatment of wet AMD. In some variations, an amount of a therapeuticagent equivalent to an amount of rapamycin of between about 1 μg andabout 5 mg is administered to the human subject; between about 20 μg andabout 1.2 mg; between about 10 μg and about 0.5 mg is administered to ahuman subject for treatment of wet AMD, between about 10 μg and 90 μg,between about 60 μg and 120 μg is administered to the human subject;between about 100 μg and 400 μg, between about 400 μg and 1 mg isadministered to the human subject is administered to the human subject;in some variations, an amount of a therapeutic agent equivalent to anamount of rapamycin of between about 1 mg and 5 mg is administered tothe human subject; in some variations, an amount of a therapeutic agentequivalent to an amount of rapamycin of between about 3 mg and 7 mg isadministered to the human subject; in some variations, an amount of atherapeutic agent equivalent to an amount of rapamycin of between about5 mg and 10 mg is administered to the human subject.

In some variations, a liquid formulation as described herein containingan amount of a therapeutic agent equivalent to an amount of rapamycin ofbetween about 1 μg and about 5 mg is administered to a human subject fortreatment of dry AMD. In some variations, an amount of a therapeuticagent equivalent to an amount of rapamycin of between about 20 μg andabout 4 mg is administered to the human subject; between about 20 μg andabout 1.2 mg; between about 10 μg and about 0.5 mg is administered to ahuman subject for treatment of wet AMD, between about 10 μg and 90 μg,between about 60 μg and 120 μg is administered to the human subject;between about 100 μg and 400 μg, between about 400 μg and 1 mg isadministered to the human subject; in some variations, an amount of atherapeutic agent equivalent to an amount of rapamycin of between about400 μg and 1 mg is administered to the human subject; in somevariations, an amount of a therapeutic agent equivalent to an amount ofrapamycin of between about 1 mg and 5 mg is administered to the humansubject; in some variations, an amount of a therapeutic agent equivalentto an amount of rapamycin of between about 3 mg and 7 mg is administeredto the human subject; in some variations, an amount of a therapeuticagent equivalent to an amount of rapamycin of between about 5 mg and 10mg is administered to the human subject to treat dry AMD.

In some variations, a liquid formulation as described herein containingan amount of a therapeutic agent equivalent to an amount of rapamycin ofbetween about 1 μg and about 5 mg is administered to a human subject forprevention of wet AMD. In some variations, an amount of a therapeuticagent equivalent to an amount of rapamycin of between about 20 μg andabout 4 mg is administered to the human subject; between about 20 μg andabout 1.2 mg; between about 10 μg and about 0.5 mg is administered to ahuman subject for prevention of wet AMD, between about 10 μg and 90 μg,between about 60 μg and 120 μg is administered to the human subject;between about 100 μg and 400 μg, between about 400 μg and 1 mg isadministered to the human subject; in some variations, an amount of atherapeutic agent equivalent to an amount of rapamycin of between about400 μg and 1 mg is administered to the human subject; in somevariations, an amount of a therapeutic agent equivalent to an amount ofrapamycin of between about 1 mg and 5 mg is administered to the humansubject; in some variations, an amount of a therapeutic agent equivalentto an amount of rapamycin of between about 3 mg and 7 mg is administeredto the human subject; in some variations, an amount of a therapeuticagent equivalent to an amount of rapamycin of between about 5 mg and 10mg is administered to the human subject to prevent wet AMD.

In some variations, any one or more of the formulations described hereinare administered intravitreally every 3 or more months, every 6 or moremonths, every 9 or more months, or every 12 or more months, or longer,to treat one or more of choroidal neovascularization, wet AMD, dry AMD,to prevent wet AMD, or to prevent progression of dry AMD to wet AMD. Insome variations, any one or more of the formulations described hereinare administered subconjunctivally every 3 or more months, every 6 ormore months, every 9 or more months, or every 12 or more months, orlonger, to treat one or more of choroidal neovascularization, wet AMD,dry AMD, or to prevent wet AMD.

In some variations, any one or more of the rapamycin formulationsdescribed herein are administered intravitreally every 3 or more months,every 6 or more months, every 9 or more months, or every 12 or moremonths, or longer, to treat one or more of choroidal neovascularization,wet AMD, dry AMD, to prevent wet AMD, or to prevent progression of dryAMD to wet AMD. In some variations, any one or more of the rapamycinformulations described herein are administered subconjunctivally every 3or more months, every 6 or more months, every 9 or more months, or every12 or more months, or longer, to treat one or more of choroidalneovascularization, wet AMD, dry AMD, or to prevent wet AMD. In somevariations, the effect of the rapamycin persists beyond the periodduring which it is present in the ocular tissues.

Delivery of the therapeutic agents described herein may, for example, bedelivered at a dosage range between about 1 ng/day and about 100 μg/day,or at dosages higher or lower than this range, depending on the routeand duration of administration. In some variations of liquid formulationor composition used in the methods described herein, the therapeuticagents are delivered at a dosage range of between about 0.1 ag/day andabout 10 μg/day. In some variations of liquid formulation or compositionused in the methods described herein, the therapeutic agents aredelivered at a dosage range of between about 1 ag/day and about 5ag/day. Dosages of various therapeutic agents for treatment, prevention,inhibition, delay of onset, or cause of regression of various diseasesand conditions described herein can be refined by the use of clinicaltrials.

The liquid formulations, including but not limited to solutions,suspensions, emulsions and situ gelling formulations, and compositionsdescribed herein may be used for delivery to the eye, as one nonlimitingexample by ocular or periocular administration, of therapeuticallyeffective amounts of rapamycin for extended periods of time to treat,prevent, inhibit, delay the onset of, or cause regression of CNV, andthus may be used to treat, prevent, inhibit, delay the onset of, orcause regression of wet AMD, or transition of dry AMD to wet AMD. It isbelieved that by changing certain characteristics of the liquidformulations described herein, including but not limited to thecomponents of the liquid formulations, the location in the eye to whichthe liquid formulation is delivered, including without limitationsubconjunctival or intravitreal placement, the liquid formulations maybe used to deliver therapeutically effective amounts of rapamycin to theeye for a variety of extended time periods including delivery oftherapeutic amounts for greater than about 1 week, for greater thanabout 2 weeks, for greater than about 3 weeks, for greater than about 1month, for greater than about 3 months, for greater than about 6 months,for greater than about 9 months, for greater than about 1 year.

When a therapeutically effective amount of rapamycin is administered toa subject suffering from wet AMD, the rapamycin may treat, inhibit, orcause regression of the wet AMD. Different therapeutically effectiveamounts may be required for treatment, inhibition or causing regression.A subject suffering from wet AMD may have CNV lesions, and it isbelieved that administration of a therapeutically effective amount ofrapamycin may have a variety of effects, including but not limited tocausing regression of the CNV lesions, stabilizing the CNV lesion, andpreventing progression of an active CNV lesion.

When a therapeutically effective amount of rapamycin is administered toa subject suffering from dry AMD, it is believed that the rapamycin mayprevent or slow the progression of dry AMD to wet AMD.

EXAMPLES

Unless the context indicates otherwise, the error bars in the chartsshow one standard deviation. Where ethanol is used, it is 200 proofethanol from Gold Shield Distributors, Hayward, Calif. Where rapamycinis used, it is from LC laboratories, Woburn, Mass., or Chunghwa ChemicalSynthesis & Biotech Co., LTD (CCSB), Taipei Hsien, Taiwan, ROC. WherePEG 400 is used, it is from The Dow Chemical Company, New Milford, Conn.Some of the graphs use the expression “uL” or “ug” to refer to μL or μg,respectively. Where a volume of 10 μL or less is administered, HamiltonHPLC syringes were used.

Example 1—Preparation and Characterization of a Rapamycin-ContainingSolution

1.256% rapamycin (percentage of the total weight) was dissolved in9.676% ethanol (percentage of the total weight). An aqueous solution of15% F127 (Lutrol) in sterile water was slowly added under continuousagitation. The final concentration was approximately 78.57% sterilewater (percentage of the total weight) and approximately 10.50% F127(Lutrol) (percentage of the total weight). This solution is listed asformulation #32 in Table 1. The solution was placed at 2° C. until use.

Example 2—Subconjunctival Injection of a Rapamycin-Containing Solution

50 μl of the solution described in Example 1 was injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits.

FIG. 2 depicts the average concentration of rapamycin present in thevitreous (ng/ml), retina choroid (ng/mg), and sclera (ng/mg) on alogarithmic scale at 20, 40, 67, and 90 days after injection.

The analysis was by liquid chromatography mass spectroscopy (LCMS) usingan internal standard.

At each timepoint, the average concentration of rapamycin was calculatedby adding the concentrations of rapamycin obtained for each eye fromeach rabbit, and dividing the total by the number of eyes analyzed. Inthis experiment, each timepoint represents the average of either twoeyes of each of two rabbits (four eyes at that timepoint) or the averageof two eyes of one rabbits (two eyes at that timepoint).

The full vitreous was homogenized and analyzed. The averageconcentration of the vitreous was calculated by dividing the mass ofrapamycin measured by the volume of vitreous analyzed. The sample didnot include the site of administration; thus, this measurement indicatedthe level of rapamycin delivered to the vitreous via the solution.

The average level of rapamycin in the vitreous at 20, 40, 67, and 90days after subconjunctival injection was about 4.425, 3.800, 4.100, and1.500 ng/ml, respectively.

The full retina choroid was homogenized and analyzed. The averageconcentration of the retina choroid was calculated by dividing the massof rapamycin measured by the mass of retina choroid analyzed. The sampledid not include the site of administration; thus, this measurementindicated the level of rapamycin delivered to the retina choroid via thesolution.

The average level of rapamycin in the retina choroid at 20, 40, 67, and90 days after subconjunctival injection was about 0.055, 0.209, 0.080,and 0.017 ng/mg, respectively.

The sclera was analyzed in the same way as the retina choroid. Thescleral sample included the site of injection; thus, this measurementindicated clearance of rapamycin from the sclera.

The average level of rapamycin in the sclera at 20, 40, 67, and 90 daysafter subconjunctival injection was about 0.141, 0.271, 0.067, and 0.192ng/mg, respectively.

Example 3—Preparation and Characterization of a Rapamycin-ContainingSolution

5.233% rapamycin (per weight of the total of the formulation after allcomponents were added) was dissolved in 0.4177 g of EtOH; the quantityof EtOH was reduced by forced evaporation (heat) to 0.1296 g (6.344%,w/w). PEG 400 was added under continuous agitation. Final concentrationsas a percentage of the total weight were approximately: rapamycin5.233%, ethanol 6.344%, and PEG 400 88.424%. When contacted with thevitreous, the formulation formed a non-dispersed mass relative to thesurrounding medium. This solution is listed as formulation #34 in Table1.

Example 4—Subconjunctival Injection of a Rapamycin-Containing Solution

25 μl of the solution described in Example 3 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits.

FIG. 3 depicts the level of rapamycin present in the vitreous (ng/ml),retina choroid (ng/mg), and sclera (ng/mg) on a logarithmic scale at 14,35, 62, and 85 days after injection. The level of rapamycin present inthe vitreous (ng/ml) is also shown at 2 days after injection.

The vitreous was homogenized and analyzed as described in Example 2,except on day 2 a single eye of each of three rabbits was analyzed; atday 14 two eyes from each of two rabbits were analyzed; at day 35 twoeyes from a single rabbit were analyzed; at day 62 two eyes from asingle rabbit were analyzed; and at day 85 one eye from a single rabbitplus two eyes from a second rabbit were analyzed.

The vitreous sample did not include the site of administration; thus,this measurement indicated the level of rapamycin delivered to thevitreous via the solution. The average level of rapamycin in thevitreous at 2, 14, 35, 62, and 85 days after subconjunctival injectionwas about 3.57, 53.65, 9.00, 4.700, and 0.600 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken on the days as described for the vitreousabove. No day 2 analysis was done. The retina choroid sample did notinclude the site of administration; thus, this measurement indicated thelevel of rapamycin delivered to the retina choroid via the solution. Theaverage level of rapamycin in the retina choroid at 14, 35, 62, and 85days after subconjunctival injection was about 0.4815, 1.725, 0.057, and0.009 ng/mg, respectively.

The scleral sample was analyzed as described in Example 2, and thesamples were taken on the days as described for the retina choroid asabove. The scleral sample included the site of injection; thus, thismeasurement indicated clearance of rapamycin from the sclera. Theaverage level of rapamycin in the sclera at 14, 35, 62, and 85 daysafter subconjunctival injection was about 34.5815, 0.135, 0.042, and0.163666667 ng/mg, respectively.

Example 5—Intravitreal Injection of a Rapamycin-Containing Solution

25 μl of the solution described in Example 3 was injected into thevitreous of the eye of New Zealand white rabbits. FIG. 4 depicts thelevel of rapamycin present in the vitreous (ng/ml), retina choroid(ng/mg), and sclera (ng/mg) on a logarithmic scale at 14, 35, 62, and 90days after injection. The level of rapamycin present in the vitreous(ng/ml) is also shown at 2 days after injection.

The vitreous was homogenized and analyzed as described in Example 2,except on day 2 approximately 1 μl of a single eye of each of threerabbits was analyzed; at day 14 two eyes from each of two rabbits wereanalyzed; at day 35 two eyes from a single rabbit were analyzed; at day62 two eyes from a single rabbit were analyzed; and at day 90 two eyesfrom each of two rabbits were analyzed.

Excepting the day 2 sample, the vitreous samples included the site ofadministration. An effort was made to avoid the administered solutionwhere possible. However, the accuracy of the measured levels ofrapamycin was potentially affected by sampling errors due to inadvertentinclusion of the administered solution.

The average level of rapamycin in the vitreous at 2, 14, 35, 62, and 90days after intravitreal injection was about 11.4, 136538, 2850.3,21820.35, and 27142.75 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken on the days described for the vitreous above.No day 2 analysis was done. The retina choroid sample did not includethe site of administration; thus, this measurement indicated the levelof rapamycin delivered to the retina choroid via the solution. Theaverage level of rapamycin in the retina choroid at 14, 35, 62, and 90days after intravitreal injection was about 5.78975, 244.485, 0.105, and1.782 ng/mg, respectively.

The scleral sample was analyzed as described in Example 2, and thesamples were taken on the days as described for the retina choroidabove. The scleral sample did not include the site of injection; thus,this measurement indicated level of rapamycin delivered to the sclera.The average level of rapamycin in the sclera at 14, 35, 62, and 90 daysafter intravitreal injection was about 0.5695, 12.34, 0.8505, and0.71175 ng/mg, respectively.

Example 6—Preparation and Characterization of a Rapamycin-ContainingSuspension

6% rapamycin (percentage of the total weight) was dispersed in 94%PEG400 (percentage of the total weight). This suspension is listed asformulation #55 in Table 1.

Example 7—Intravitreal Injection of a Rapamycin-Containing Suspension

The solution prepared in Example 6 was injected intravitreally into theeyes of New Zealand white rabbits. FIG. 5 depicts images of rabbit eyesafter intravitreal injection of 10 μl (FIG. 5A), 20 μl (FIG. 5B), and 40μl (FIG. 5C) of a 6% rapamycin suspension in PEG400. This resulted in aninjected dose of about 0.6, about 1.2, and about 2.4 mg. The images werefocused on the administered suspension. These images show that thesuspension forms a non-dispersed mass relative to the surroundingvitreal medium.

Example 8—Preparation and Characterization of a Rapamycin-Containing InSitu Gelling Formulation

A liquid formulation of 4.2% rapamycin (obtained from LC laboratories inWoburn, Mass., and Chunghwa Chemical Synthesis & BioTech. Co, Ltd inTaiwan), 4.3% ethanol (obtained from Gold Shield Chemical in Hayward,Calif.), 2.2% PVP K90 (obtained from BASF), 87.1% PEG 400 (obtained fromDOW Chemical), and 2.2% Eudragit RL 100 (obtained from Rohm PharmaPolymers), where all percentages are by weight of the total.

Eudragit RL 100 was dissolved in ethanol. Sonication and heat may berequired at this step. Ethanol—Eudragit was added to PEG 400. PVP wasslowly added to the Eudragit-Ethanol-PEG solution, and a uniformly mixedsolution was obtained. Vigorous mixing may be required for this step.

Rapamycin was added to and dissolved in the Eudragit-ethanol-PEG-PVPmix. Heat and sonication may be used. The formulation was mixedthoroughly (using a vortex or mixer) to achieve uniformity. Thisformulation is listed as #37 in Table 1.

When placed in deionized water or tap water, the liquid formulationformed a non-dispersed mass. The non-dispersed mass appeared as agel-like substance.

Example 9—Subconjunctival Injection of a Rapamycin-ContainingNon-Dispersed Mass-Forming Formulation

50 μl of the solution described in Example 8 was injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits.

FIG. 6 depicts the average concentration of rapamycin present in thevitreous (ng/ml), retina choroid tissues (ng/mg), and sclera (ng/mg) ona logarithmic scale at 7, 32, 45, and 90 days after injection of the insitu gelling formulation.

The analysis was by LCMS (liquid chromatography-mass spectroscopy).

Where more than a single eye was analyzed, the average concentration ofrapamycin was calculated by adding the concentrations of rapamycinobtained for each eye from each rabbit, and dividing the total by thenumber of eyes analyzed. In this experiment, the vitreous day 7 and thesclera day 7, 32, and 45 timepoints represent a single eye, as opposedto an average level. The remaining day 7, 32, and 45 timepointsrepresent the average of two eyes of one rabbit, and the day 90timepoint represents the average of two eyes of each of two rabbits(four eyes total).

The full vitreous was homogenized and analyzed. The averageconcentration of the vitreous was calculated by dividing the mass ofrapamycin measured by the volume of vitreous analyzed. The sample didnot include the site of administration; thus, this measurement indicatedthe level of rapamycin delivered to the vitreous via the in situ gellingformulation.

The average level of rapamycin in the vitreous at 7, 32, 45, and 90 daysafter subconjunctival injection was about 13.9, about 7.4, about 1.35,and about 9.9 ng/ml, respectively.

The full retina choroid tissues were homogenized and analyzed. Theaverage concentration of the retina choroid tissues was calculated bydividing the mass of rapamycin measured by the mass of retina choroidtissues analyzed. The sample did not include the site of administration;thus, this measurement indicated the level of rapamycin delivered to theretina choroid tissues via the in situ gelling formulation.

The average level of rapamycin in the retina choroid tissues at 7, 32,45, and 90 days after subconjunctival injection was about 0.376, about0.1875, about 0.136, and about 0.29 ng/mg, respectively.

The sclera was analyzed in the same way as the retina choroid tissues.The scleral sample may have included the injected liquid formulation;thus, this measurement was indicative of clearance of rapamycin from thesclera.

The average level of rapamycin in the sclera at 7, 32, 45, and 90 daysafter subconjunctival injection was about 2033, about 1653, about 3626,and about 420.5 ng/mg, respectively.

Example 10—Preparation and Characterization of a Rapamycin-ContainingSuspension

A rapamycin containing suspension was formed by dispersing 150.5 mg ofrapamycin (3.004% by weight) in 4860.3 mg of PEG 400 (96.996% byweight). This formulation is listed as #49 in Table 1.150.5 mg rapamycin(3.004% by weight) and 4860.3 mg of PEG 400 (96.996% by weight) wereplaced in an amber vial. High Wear Resistant Zirconia Grinding Media(beads) of 3 mm diameter were added, up to three quarters of the totalvolume. The vial was sealed and placed in a Cole-Parmer millingapparatus for 48 hrs. The particle size median for rapamycin was 2.8386mm and the mean was 3.1275 mm. The formulation was kept at 4 C untiluse. Volumes of 20 μl and 40 μl each formed a non-dispersed mass whenplaced in the vitreous of a rabbit eye.

Example 11—Subconjunctival Injection of a Rapamycin-ContainingSuspension

40 μl of the suspension described in Example 10 were injected betweenthe sclera and the conjunctiva of the eye of New Zealand white rabbits.FIG. 7 depicts the level of rapamycin in the vitreous (ng/ml), retinachoroid (ng/mg), and the sclera (ng/mg) on a logarithmic scale at 14,42, 63, and 91 days after injection.

The vitreous was homogenized and analyzed as described in Example 2. Twoeyes from each of two rabbits were analyzed at each time point exceptfor day 91, on which two eyes from one rabbit were analyzed. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 14, 42, 63, and 91days after subconjunctival injection was about 4.031, 23.11, 53.27, and13.94 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 14, 42,63, and 91 days after subconjunctival injection was about 0.1577, 4.965,0.385, and 0.05 ng/mg, respectively.

The scleral sample was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Thescleral sample included the site of injection. The average level ofrapamycin in the sclera at 14, 42, 63, and 91 days after subconjunctivalinjection was about 1283, 476.3, 854.2, and 168.5 ng/mg, respectively.

Example 12—Intravitreal Injection of a Rapamycin-Containing Suspension

20 μl of the suspension described in Example 10 were injected into thevitreous of the eye of New Zealand white rabbits. The injectedsuspension formed a non-dispersed mass relative to the surroundingmedium. FIG. 8 depicts the level of rapamycin in the retina choroid(ng/mg) and the sclera (ng/mg) on a logarithmic scale at 14, 42, 63, and91 days after injection and in the vitreous (ng/ml) at 63 and 91 daysafter injection.

The vitreous was homogenized and analyzed as described in Example 2. Twoeyes from each of two rabbits were analyzed at each time point. Thevitreous sample may have included the site of administration. Theaverage level of rapamycin in the vitreous at 63 and 91 days afterintravitreal injection was about 381,600 and 150,400 ng/ml,respectively.

The retina choroid was homogenized and analyzed as described in Example2. Two eyes from each of two rabbits were analyzed at each time point.The retina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 14, 42,63, and 91 days after intravitreal injection was about 2.588, 4.249,21.42, and 0.922 ng/mg, respectively.

The scleral sample was homogenized and analyzed as described in Example2, with the samples taken as described for the retina choroid above. Thescleral sample did not include the site of injection, so thismeasurement indicated the level of rapamycin delivered to the sclera.The average level of rapamycin in the sclera at 14, 42, 63, and 91 daysafter intravitreal injection was about 0.7327, 6.053, 1.373, and 17.49ng/mg, respectively.

Example 13—Preparation and Characterization of a Rapamycin-ContainingSolution

A rapamycin containing solution was formed by placing 116.6 mg ofrapamycin in ethanol and storing the mixture at 4° C. for 6 hours. Thissolution was then mixed with 4647.5 mg of PEG 400 to give a solutionhaving final concentrations by weight of 2.29% rapamycin, 6.05% ethanol,and 91.66% PEG 400. This solution is listed as formulation #51 inTable 1. A volume of 30 μl formed a non-dispersed mass when placed inthe vitreous of rabbit eyes.

Example 14—Subconjunctival Injection of a Rapamycin-Containing Solution

40 μl of the solution described in Example 13 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.9 depicts the level of rapamycin in the vitreous (ng/ml), retina choroid(ng/mg), and the sclera (ng/mg) on a linear scale at 14, 42, 63, and 91days after injection.

The vitreous was homogenized and analyzed as described in Example 2. Twoeyes from each of two rabbits were analyzed at each time point exceptfor day 91, on which two eyes from one rabbit were analyzed. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 14, 42, 63, and 91days after subconjunctival injection was about 1.804, 1.854, 1.785, and1.255 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 14, 42,63, and 91 days after subconjunctival injection was about 1.221, 4.697,0.1075, and 0.02 ng/mg, respectively.

The scleral sample was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Thescleral sample included the site of injection. The average level ofrapamycin in the sclera at 14, 42, 63, and 91 days after subconjunctivalinjection was about 1.987, 1.884, 0.56, and 10.84 ng/mg, respectively.

Example 15—Intravitreal Injection of a Rapamycin-Containing Solution

30 μl of the solution described in Example 13 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 10depicts the level of rapamycin in the retina choroid (ng/mg) and thesclera (ng/mg) on a linear scale at 14, 42, 63, and 91 days afterinjection.

The retina choroid was homogenized and analyzed as described in Example2. Two eyes from each of two rabbits were analyzed at each time point.The retina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 14, 42,63, and 91 days after intravitreal injection was about 5.515, 5.388,0.3833, and 11.52 ng/mg, respectively.

The scleral sample was homogenized and analyzed as described in Example2, with the samples taken as described for the retina choroid above. Thescleral sample did not include the site of injection, so thismeasurement indicated the level of rapamycin delivered to the sclera.The average level of rapamycin in the sclera at 14, 42, 63, and 91 daysafter intravitreal injection was about 1.077, 0.9239, 0.0975, and 2.0825ng/mg, respectively.

FIG. 11 depicts the level of rapamycin in the vitreous (ng/ml) on alinear scale at 63 and 91 days after injection. The vitreous washomogenized and analyzed as described in Example 2. Two eyes from eachof two rabbits were analyzed at each time point. The vitreous sample mayhave included the site of administration. The average level of rapamycinin the vitreous at 63 and 91 days after intravitreal injection was about299,900 and 196,600 ng/ml, respectively.

Example 16—Preparation and Characterization of a Rapamycin-ContainingSolution

About 320 g of ethanol was sparged with N₂ for about 10 minutes, andthen about 40 g of sirolimus was added to the ethanol. The mixture wassonicated for about 20 minutes, by the end of which all of the sirolimushad gone into solution to form a sirolimus stock solution. A diluentsolvent was prepared by sonicating about 1880 g of PEG 400 for about 60minutes, and then sparging the solvent with Nitrogen for about 10minutes.

The sirolimus stock solution and the PEG 400 were then rotated at aboutroom temperature in a rotary evaporator for about 10 minutes to mix thestock solution with the diluent solvent. After mixing, the solution wassparged with nitrogen for about 10 minutes and blanketed with nitrogenfor about 5 minutes. After the solution was sparged and filled withnitrogen, about 240 g of excess ethanol was evaporated from the solutionby increasing the solution temperature, maintaining a temperature thatdid not exceed 40° C. for an extended period of time and continuing torotate the solution for about 2.5 hours.

The resulting solution comprised about 40 g of sirolimus (about 2% byweight), about 80 g of ethanol (about 4% by weight), and about 1880 g ofPEG 400 (about 94% by weight). This solution was sparged with nitrogenfor about 10 minutes and blanketed with nitrogen for about 5 minutes.The solution was then filtered through a 0.2 micron filter. HPLC vialswere filled with 2 ml each of the filtered solution to leave a headspace in each container of about 400 μl. This head space was filled withnitrogen gas and capped.

Example 17—Preparation and Characterization of a Rapamycin-ContainingSolution

Rapamycin, ethanol and PEG 400 were placed in a container to give finalconcentrations by weight of about 2.00% rapamycin, about 4.00% ethanol,and about 94.00% PEG 400. The mixture was capped and sonicated for 1-2hours. The sonication generated heat, with temperatures of up to about40 or 50° C. This solution is listed as formulation #100 in Table 1.Volumes of 1 μl, 3 μl, 20 μl, and 40 μl formed a non-dispersed mass inthe vitreous of rabbit eyes.

Example 18—Subconjunctival Injection of a Rapamycin-Containing Solution

20 μl of the solution described in Example 17 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.12 depicts the level of rapamycin in the vitreous on a logarithmic scaleat 5, 30, 60, 90, and 120 days after injection. FIG. 13 depicts thelevel of rapamycin in the retina choroid on a logarithmic scale at thesame time points. For comparison, FIG. 12 and FIG. 13 also depictresults of similar studies, performed with 40 μl and 60 μl injections,described below in Example 19 and Example 20.

In FIGS. 12-15, discussed in this and following examples, some outlierpoints have been omitted. Individual data points from the same study atthe same time point were compared to each other. When the arithmeticmean of the data points was lower than their standard deviation, thedata points that were higher or lower by an order of magnitude wereconsidered as outliers.

The vitreous was homogenized and analyzed as described in Example 2.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after subconjunctival injection was about 1.81, 0.45, 0.39, 1.85,and 1.49 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after subconjunctival injection was about 0.14,0.03, 0.02, 0.02, and 0.01 ng/mg, respectively.

Example 19—Subconjunctival Injection of a Rapamycin-Containing Solution

40 μl of the solution described in Example 17 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.12 depicts the level of rapamycin in the vitreous on a logarithmic scaleat 5, 30, 60, 90, and 120 days after injection. FIG. 13 depicts thelevel of rapamycin in the retina choroid on a logarithmic scale at thesame time points.

The vitreous was homogenized and analyzed as described in Example 2.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after subconjunctival injection was about 2.39, 0.65, 0.54, 2.07,and 1.92 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after subconjunctival injection was about 0.47,0.04, 0.01, 0.05, and 0.0 ng/mg, respectively.

Example 20—Subconjunctival Injection of a Rapamycin-Containing Solution

60 μl of the solution described in Example 17 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.12 depicts the level of rapamycin in the vitreous on a logarithmic scaleat 5, 30, 60, 90, and 120 days after injection. FIG. 13 depicts thelevel of rapamycin in the retina choroid on a logarithmic scale at thesame time points.

The vitreous was homogenized and analyzed as described in Example 2.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after subconjunctival injection was about 8.65, 0.29, 0.18, 2.00,1.41 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after subconjunctival injection was about 0.63,0.02, 0.02, 0.06, and 0.01 ng/mg, respectively.

Example 21—Intravitreal Injection of a Rapamycin-Containing Solution

20 μl of the solution described in Example 17 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 14depicts the level of rapamycin in the vitreous on a logarithmic scale 5,30, 60, 90, and 120 days after injection. FIG. 15 depicts the level ofrapamycin in the retina choroid on a logarithmic scale at the same timepoints. For comparison, FIG. 14 and FIG. 15 also depict results of otherstudies described below in Example 22 and Example 24.

The vitreous was homogenized and analyzed as described in Example 2.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample may have included the site of administration. Theaverage level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after intravitreal injection was about 162,100; 18,780; 57,830;94,040; and 13,150 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after intravitreal injection was about 2.84, 2.26,0.17, 0.22, and 0.05 ng/mg, respectively.

Example 22—Intravitreal Injection of a Rapamycin-Containing Solution

40 μl of the solution described in Example 17 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 14depicts the level of rapamycin in the vitreous on a logarithmic scale 5,30, 60, 90, and 120 days after injection. FIG. 15 depicts the level ofrapamycin in the retina choroid on a logarithmic scale at the same timepoints.

The vitreous was homogenized and analyzed as described in Example 2.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample may have included the site of administration. Theaverage level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after intravitreal injection was about 415,600; 4,830; 74,510;301,300; and 7,854 ng/ml respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after intravitreal injection was about 5.36, 0.23,1.27, 1.08, and 0.08 ng/mg, respectively.

Example 23—Preparation and Characterization of a Rapamycin-ContainingSolution

Rapamycin, ethanol and PEG 400 were added to a container to give finalconcentrations by weight of about 0.4% rapamycin, 4.0% ethanol, and95.6% PEG 400. The mixture was sonicated for 1-2 hours. Sonicationresulted in elevated temperatures of up to about 40 to 50° C. Thissolution is listed as formulation #99 in Table 1.

Example 24—Intravitreal Injection of a Rapamycin-Containing Solution

100 μl of the solution described in Example 23 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutiondid not form a non-dispersed mass relative to the surrounding medium.FIG. 14 depicts the level of rapamycin in the vitreous on a logarithmicscale at 5, 30, 60, 90, and 120 days after injection. FIG. 15 depictsthe level of rapamycin in the retina choroid on a logarithmic scale atthe same time points.

The vitreous was homogenized and analyzed as described in Example 2.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample may have included the site of administration. Theaverage level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after intravitreal injection was about 151,000; 14,890; 4,743; and1620 ng/ml respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after intravitreal injection was about 1.21, 1.84,0.04, 0.71, and 0.0 ng/mg, respectively.

Example 25—Preparation and Characterization of a Rapamycin-ContainingSolution

A rapamycin containing solution was formed by placing 102.4 mg ofrapamycin in ethanol, adding 4719.3 mg of PEG 400, and vortexing. Theresulting solution had final concentrations by weight of 2.036%rapamycin, 4.154%% ethanol, and 93.81% PEG 400. This solution is listedas formulation #139 in Table 1.

Example 26—Subconjunctival Injection of a Rapamycin-Containing Solution

10 μl of the solution described in Example 25 were injected as a singledose between the sclera and the conjunctiva of the eye of New Zealandwhite rabbits. FIG. 16 depicts the level of rapamycin in the vitreous ona logarithmic scale at 5 and 14 days after injection. FIG. 17 depictsthe level of rapamycin in the retina choroid on a logarithmic scale atthe same time points. For comparison, FIG. 16 and FIG. 17 also depictresults of other studies described below in Examples 27-29.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5 and 14 days aftersubconjunctival injection was about 2.45 and 20.13 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5 and14 days after subconjunctival injection was about 0.13 and 0.19 ng/mg,respectively.

Example 27—Subconjunctival Injection of a Rapamycin-Containing Solution

60 μl of the solution described in Example 25 were injected as a singledose between the sclera and the conjunctiva of the eye of New Zealandwhite rabbits. FIG. 16 depicts the level of rapamycin in the vitreous ona logarithmic scale at 5 and 14 days after injection. FIG. 17 depictsthe level of rapamycin in the retina choroid on a logarithmic scale atthe same time points.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5 and 14 days aftersubconjunctival injection was about 17.98 and 87.03 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5 and14 days after subconjunctival injection was about 0.27 and 0.21 ng/mg,respectively.

Example 28—Subconjunctival Injection of a Rapamycin-Containing Solution

60 μl of the solution described in Example 25 were injected as two 30 μldoses at two sites between the sclera and the conjunctiva of the eye ofNew Zealand white rabbits. FIG. 16 depicts the level of rapamycin in thevitreous on a logarithmic scale at 5 and 14 days after injection. FIG.17 depicts the level of rapamycin in the retina choroid on a logarithmicscale at the same time points.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5 and 14 days aftersubconjunctival injection was about 502.2 and 31.80 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5 and14 days after subconjunctival injection was about 0.80 and 0.15 ng/mg,respectively.

Example 29—Subconjunctival Injection of a Rapamycin-Containing Solution

90 μl of the solution described in Example 25 were injected as three 30μl doses at three sites between the sclera and the conjunctiva of theeye of New Zealand white rabbits. FIG. 16 depicts the level of rapamycinin the vitreous on a logarithmic scale at 5 and 14 days after injection.FIG. 17 depicts the level of rapamycin in the retina choroid on alogarithmic scale at the same time points.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5 and 14 days aftersubconjunctival injection was about 39.05 and 13.63 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5 and14 days after subconjunctival injection was about 0.83 and 0.10 ng/mg,respectively.

Example 30—Preparation and Characterization of a Rapamycin-ContainingSuspension

A rapamycin containing suspension was formed by placing 201.6 mg ofrapamycin (3.000% by weight) in 6518.8 mg of PEG 400 (97.000% by weight)and vortexing. The resulting particle size was not quantified but it waslarge, estimated at about 10 μm. This suspension is listed asformulation #147 in Table 1.

Example 31—Subconjunctival Injection of a Rapamycin-ContainingSuspension

10 μl of the suspension described in Example 30 were injected as asingle dose between the sclera and the conjunctiva of the eye of NewZealand white rabbits. FIG. 18 depicts the level of rapamycin in thevitreous on a logarithmic scale at 5, 14, and 30 days after injection.FIG. 19 depicts the level of rapamycin in the retina choroid on alogarithmic scale at the same time points. For comparison, FIG. 18 andFIG. 19 also depict results of other studies described below in Example32 and Example 33.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5, 14, and 30 days aftersubconjunctival injection was about 2.68, 0.90, and 5.43 ng/ml,respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 14,and 30 days after subconjunctival injection was about 0.20, 0.06, and1.23 ng/mg, respectively.

Example 32—Subconjunctival Injection of a Rapamycin-ContainingSuspension

30 μl of the solution described in Example 30 were injected as a singledose between the sclera and the conjunctiva of the eye of New Zealandwhite rabbits. FIG. 18 depicts the level of rapamycin in the vitreous ona logarithmic scale at 5, 14, and 30 days after injection. FIG. 19depicts the level of rapamycin in the retina choroid on a logarithmicscale at the same time points.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5, 14, and 30 days aftersubconjunctival injection was about 84.55, 11.23, and 66.35 ng/ml,respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 14,and 30 days after subconjunctival injection was about 1.09, 0.19, and1.02 ng/mg, respectively.

Example 33—Subconjunctival Injection of a Rapamycin-ContainingSuspension

90 μl of the solution described in Example 30 were injected as three 30μl doses at three sites between the sclera and the conjunctiva of theeye of New Zealand white rabbits. FIG. 18 depicts the level of rapamycinin the vitreous on a logarithmic scale at 5, 14, and 30 days afterinjection. FIG. 19 depicts the level of rapamycin in the retina choroidon a logarithmic scale at the same time points.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous sampledid not include the site of administration, so this measurementindicated the level of rapamycin delivered to the vitreous. The averagelevel of rapamycin in the vitreous at 5, 14, and 30 days aftersubconjunctival injection was about 29.95, 15.30, and 49.20 ng/ml,respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 14,and 30 days after subconjunctival injection was about 0.55, 1.31, and5.74 ng/mg, respectively.

Example 34—Preparation and Characterization of a Rapamycin-ContainingSolution

10.3 mg of rapamycin was placed in ethanol, 4995.8 mg of PEG 400 wasadded, and the mixture was vortexed to give a solution having finalconcentrations by weight of 0.205% rapamycin, 0.544% ethanol, and99.251% PEG 400. This solution is listed as formulation #140 in Table 1.A volume of 10 μl of this solution formed a non-dispersed mass whenplaced in the vitreous of a rabbit eye.

Example 35—Intravitreal Injection of a Rapamycin-Containing Solution

10 μl of the solution described in Example 34 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 20depicts the level of rapamycin in the retina choroid on a logarithmicscale at 5 and 30 days after injection. FIG. 21 depicts the level ofrapamycin in the vitreous on a logarithmic scale at the same timepoints.For comparison, FIG. 20 and FIG. 21 also depict results of other studiesdescribed below in Example 37 and Example 39.

The vitreous was homogenized and analyzed as described in Example 2.Five rabbit eyes were analyzed at each time point. The vitreous samplemay have included the site of administration. The average level ofrapamycin in the vitreous at 5 and 30 days after intravitreal injectionwas about 12.02 and 0.92 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5 and30 days after intravitreal injection was about 0.08 and 0.02 ng/mg,respectively.

Example 36—Preparation and Characterization of a Rapamycin-ContainingSolution

31.5 mg of rapamycin was placed in ethanol, 4918.9 mg of PEG 400 wasadded, and the solution was vortexed. Final concentrations by weightwere 0.6238% rapamycin, 1.337% ethanol, and 98.035% PEG 400. Thissolution is listed as formulation #142 in Table 1. The formulation wasstored at 4° C. until use. A volume of 10 μl of this solution formed anon-dispersed mass when placed in the vitreous of a rabbit eye.

Example 37—Intravitreal Injection of a Rapamycin-Containing Solution

10 μl of the solution described in Example 36 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 20depicts the level of rapamycin in the retina choroid on a logarithmicscale at 5 and 30 days after injection. FIG. 21 depicts the level ofrapamycin in the vitreous on a logarithmic scale at the same timepoints.

The vitreous was homogenized and analyzed as described in Example 2.Five rabbit eyes were analyzed at each time point. The vitreous samplemay have included the site of administration. The average level ofrapamycin in the vitreous at 5 and 30 days after intravitreal injectionwas about 87.46 and 44.34 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5 and30 days after intravitreal injection was about 1.40 and 0.01 ng/mg,respectively.

Example 38—Preparation and Characterization of a Rapamycin-ContainingSolution

103.5 mg of rapamycin was placed in ethanol, 4720.8 mg of PEG 400 wasadded, and the mixture was vortexed to give a solution having finalconcentrations by weight of 2.057% rapamycin, 4.116% ethanol, and93.827% PEG 400. This solution is listed as formulation #144 in Table 1.A volume of 10 μl of this solution formed a non-dispersed mass in thevitreous of a rabbit eye.

Example 39—Intravitreal Injection of a Rapamycin-Containing Solution

10 μl of the solution described in Example 38 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 20depicts the level of rapamycin in the retina choroid on a logarithmicscale at 5, 30, and 90 days after injection. FIG. 21 depicts the levelof rapamycin in the vitreous on a logarithmic scale at the sametimepoints.

The vitreous was homogenized and analyzed as described in Example 2.Four rabbit eyes were analyzed at each time point. The vitreous samplemay have included the site of administration. The average level ofrapamycin in the vitreous at 5, 30, and 90 days after intravitrealinjection was about 120,500; 55,160; and 0.55 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above exceptthat five rabbit eyes were analyzed at the 5 and 30 day time points. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,and 90 days after intravitreal injection was about 4.75, 0.17, and 0.01ng/mg, respectively.

Example 40—Subconjunctival Injection of a Rapamycin-Containing Solution

40 μl of the solution described in Example 17 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.22 depicts on a logarithmic scale the level of rapamycin in the aqueoushumor (ng/ml) at 1, 4, 7, 11, 14, 21, 28, 35, 54, and 56 days afterinjection, and the levels of rapamycin in the cornea (ng/mg) and theretina choroid (ng/mg) at 4, 14, 21, and 35 days after injection. Theretina choroid level is labeled as “R/Choroid” in FIG. 22.

The aqueous humor was homogenized and then analyzed by liquidchromatography and mass spectroscopy. Four rabbit eyes were analyzed foreach time point. The aqueous humor did not include the site ofinjection, so this measurement indicated the level of rapamycindelivered to the aqueous humor. The average level of rapamycin in theaqueous humor at 1, 4, 7, 11, 14, 21, 28, 35, 54, and 56 days afterinjection was about 0.875, 1.0, 7.0, 0.725, 0.5, 0.525, 0.0, 0.125,0.014, and 0.0485 ng/ml, respectively.

The cornea was homogenized and then analyzed by liquid chromatographyand mass spectroscopy. The cornea did not include the site of injection,so this measurement indicated the level of rapamycin delivered to thecornea. Four rabbit eyes were analyzed for each time point. The averagelevel of rapamycin in the cornea at 4, 14, 21, and 35 days afterinjection was about 0.3225, 0.1, 0.0275, and 0.0125 ng/mg, respectively.

The retina choroid was homogenized and analyzed as described in Example2, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 4, 14,21, and 35 days after injection was about 11.61, 0.2, 0.0275, and 2.655ng/mg, respectively.

Example 41—Intravitreal Injection of a Rapamycin-Containing Solution

1.0 μl of the solution described in Example 17 was injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. Table 2reports the average level of rapamycin in the aqueous humor one dayafter injection. For comparison, Table 2 also reports results of studiesdescribed in Examples 42-45 below.

The aqueous humor was homogenized and analyzed as described in Example40. Two rabbit eyes were analyzed. The aqueous humor did not include thesite of injection, so this measurement indicated the level of rapamycindelivered to the aqueous humor. The average level of rapamycin in theaqueous humor at 1 day after injection was about 0.438 ng/ml with astandard deviation of about 0.141 ng/ml.

Example 42—Intravitreal Injection of a Rapamycin-Containing Solution

3.0 μl of the solution described in Example 17 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. Table 2reports the average level of rapamycin in the aqueous humor one dayafter injection.

The aqueous humor was homogenized and analyzed as described in Example40. Two rabbit eyes were analyzed. The aqueous humor did not include thesite of injection, so this measurement indicated the level of rapamycindelivered to the aqueous humor. The average level of rapamycin in theaqueous humor at 1 day after injection was about 0.355 ng/ml with astandard deviation of about 0.234 mg/ml.

Example 43—Subconjunctival Injection of a Rapamycin-Containing Solution

3.0 μl of the solution described in Example 17 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. Theinjected solution formed a non-dispersed mass relative to thesurrounding medium. Table 2 reports the average level of rapamycin inthe aqueous humor one day after injection.

The aqueous humor was homogenized and analyzed as described in Example40. Two rabbit eyes were analyzed. The aqueous humor did not include thesite of injection, so this measurement indicated the level of rapamycindelivered to the aqueous humor. The average level of rapamycin in theaqueous humor at 1 day after injection was about 0.338 ng/ml with astandard deviation of about 0.122 ng/ml.

Example 44—Anterior Chamber Administration of a Rapamycin-ContainingSolution

5.0 μl of the solution described in Example 17 were injected into theanterior chamber of the eye of New Zealand white rabbits by injectioninto the front-end of the eye. The aqueous humor was withdrawn using asyringe. Table 2 reports the average level of rapamycin in the aqueoushumor 14 days after injection.

The aqueous humor was homogenized and analyzed as described in Example40. Two rabbit eyes were analyzed. The aqueous humor did not include thesite of injection, so this measurement indicated the level of rapamycindelivered to the aqueous humor. The average level of rapamycin in theaqueous humor at 14 days after injection was about 0.166 ng/ml with astandard deviation of about 0.183 ng/ml.

Example 45—Anterior Chamber Administration of a Rapamycin-ContainingSolution

10 μl of the solution described in Example 17 were injected into theanterior chamber of the eye of New Zealand white rabbits. Table 2reports the average level of rapamycin in the aqueous humor 14 daysafter injection.

The aqueous humor was homogenized and analyzed as described in Example40. Two rabbit eyes were analyzed. The aqueous humor did not include thesite of injection, so this measurement indicated the level of rapamycindelivered to the aqueous humor. The average level of rapamycin in theaqueous humor at 14 days after injection was about 0.004 ng/ml with astandard deviation of about 0.006 ng/ml.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not.

TABLE 1 Liquid Formulations Median NDM, Form. Form. particle Injection #Composition (mg), % (w/w) Type size volume  1 DMSO = 2000 mg (20%) SWater = 8000 mg (80%)  2 F68 = 1000 mg (10%) S Water = 9000 mg (90%)  3F68 = 3000 mg (30%) S Water = 7000 mg (70%)  4 F127 = 1000 mg (10%) SWater = 9000 mg (90%)  5 F127 = 1500 mg (15%) S Water = 8500 mg (85%)  6Beta-cyclodextrin = 250 mg (2.5%) S Water = 9750 mg (97.5%)  7 Rapa =10.2 mg (0.101%) S No, 50 μL Pluronic, F68 = 1010 mg (9.99%) Water =9090 mg (89.909%)  8 Rapa = 10.2 mg (0.102%) S No, 50 μL Pluronic, F68 =3000 mg (29.969%) Water = 7000 mg (69.929%)  9 Rapa = 10.5 mg (0.104%) SNo, 50 μL Pluronic, F127 = 1010 mg (9.99%) Water = 9090 mg (89.907%)  10Rapa = 10.5 mg (0.105%) S No, 50 μL Pluronic, F127 = 1500 mg (14.984%)Water = 8925 mg (84.9%)  11 Rapa = 10.7 mg (0.105%) S No, 50 μLBeta-cyclodextrin = 255 mg (2.497%) Water = 9945 mg (97.398%)  12 Rapa =6.4 mg (0.0999%) SP CMC = 48 mg (0.7493%) Polysorbitan 20 = 2.56 mg(0.04%) Water = 6349.44 mg (99.111%)  13 Rapa = 6.5 mg (0.0999%) S DMSO= 325 mg (4.995%) Water = 6175 mg (94.905%)  14 Rapa = 13.5 mg (0.0999%)SP CMC = 101.25 mg (0.7493%) Polysorbitan 20 = 5.4 mg (0.04%) Water =13393.35 mg (99.112%)  15 Rapa = 11.0 mg (0.2%) S EtOH = 5500 mg (99.8%) 16 Rapa = 6.6 mg (0.1%) S EtOH = 1054.6 mg (15.933%) F127 = 833.64 mg(12.595%) Water = 4723.96 mg (71.372%)  17 Rapa = 5 mg (0.1%) S Cavitron= 0.25 g (5%) Ethanol, 95% = 57 mg (1.1%) Sterile water = 4.753 g(93.8%)  18 Rapa = 5 mg (0.1%) S Ethanol, 95% = 150 mg (2.9%) PEG400 =1.0 g (19.4%) Sterile water = 4.01 g (77.6%)  19 Rapa = 5 mg (0.1%) SYes 50 μL Ethanol, 95% = 152 mg (3.2%) PEG400 = 1.5227 g (30.2%) Sterilewater = 3.3592 g (66.67%)  20 Rapa = 6.6 mg (0.1%) S EtOH = 505.1 mg(7.618%) F127 = 917.8 mg (13.843%) Water = 5200.6 mg (78.44%)  21 Rapa =6.6 mg (0.1%) S No, 50 μL EtOH = 536 mg (7.5%) Pluronic, F127 = 983.75mg (14.0%) Water = 5574.56 mg (78.4%)  22 Rapa = 5.2 mg (0.1023%) S EtOH= 56.6 mg (1.127%) Captisol = 2008.9 mg (39.5%) Water = 3013.3 mg(59.3%)  23 Rapa = 6.9 mg (0.201%) S EtOH = 3418.0 mg (99.799%)  24 Rapa= 9.1 mg (0.491%) S EtOH = 90.9 mg (4.908%) F127 = 262.8 mg (14.191%)Water = 1489.1 mg (80.409%)  25 Rapa = 0 mg (0%) S EtOH = 310.2 mg(5.144%) F127 = 858.1 mg (14.228%) Water = 4862.6 mg (80.628%)  26 Rapa= 0 mg (0%) S EtOH = 613.1 mg (10.19%) F127 = 810.6 mg (13.471%) Water =4593.6 mg (76.339%)  27 Rapa = 53.5 mg (1.095%) S Yes, 50 μL EtOH =414.8 mg (8.488%) F127 = 662.8 mg (13.563%) Water = 3755.7 mg (76.854%) 28 Rapa = 0.3 g (10%) ISG, SP PVP K90 = 0.35 g (12%) Eudragit RS30D =2.35 g (78%)  29 Rapa = 0.2154 g (7.31%) ISG, SP PVP K90 = 0.25 g (8.5%)Eudragit RS30D = 2.48 g (84.19%)  30 Rapa = 53.9 mg (1.103%) S No, 50 μLEtOH = 413.6 mg (8.463%) Sterile water = 3843.5 mg (78.647%) F127(Lutrol) = 576.0 mg (11.786%)  31 Rapa = 0 mg (0%) S EtOH = 411.9 mg(8.513%) Sterile Water = 3849.3 mg (79.554%) F127(Lutrol) = 577.4 mg(11.933%)  32 Rapa = 54.1 mg (1.256%) S EtOH = 416.8 mg (9.676%) SterileWater = 3836.3 mg (78.569%) F127(Lutrol) = 577.5 mg (10.499%)  33 Rapa =80.7 g (1.964%) S EtOH = 65.0 mg (0.158%) PEG400 = 4021.8 mg (97.878%) 34 Rapa = 106.9 g (5.233%) S Yes, 25 μL EtOH = 129.6 mg (6.344%) PEG400= 1806.5 mg (88.424%)  35 Rapa = 0 mg (0%) ISG, SP PVP K90 = 0.204 g(2.3%) Ethanol, 100% = 0.4 g (4.5%) Eudragit RL100 = 0.201 g (2.3%) PEG400 = 8.00 g (90.9%)  36 Rapa = 0 mg (0%) ISG, SP PVP K90 = 0.2 g (2.2%)Ethanol, 100% = 0.4 g (4.4%) PVAP = 0.4 g (4.4%) PEG 400 = 8.00 g(88.9%)  37 Rapa = 106.1 mg (4.2%) ISG, SP PVP K90 = 55.2 mg (2.2%)Ethanol, 100% = 108 mg (4.3%) Eudragit RL100 = 55 mg (2.2%) PEG 400 =2.2 g (87.1%)  38 Rapa = 399.6 mg (9.965%) S Yes, 20 μL F68(Lutrol) =40.6 mg (1.012%) Sterile Water = 3569.7 mg (89.022%)  39 Rapa = 53.8 mg(1.1%) S EtOH = 415.2 mg (8.489%) Sterile Water = 3844.2 mg (78.594%)F127 = 578.0 mg (11.817%)  40 Rapa = 208.1 mg (3.148%) S Yes, 20 μLPEG400 = 6403.4 mg (96.852%)  41 Rapa = 200.4 mg (5.148%) SP F68(Lutrol)= 20.8 mg (0.534%) PEG400 = 3569.3 mg (91.697%) EtOH (95%) = 102 mg(2.62%)  42 Rapa = 200.4 g (5.259%) SP PEG400 = 3561.4 mg (93.46%) Tween80 = 48.8 mg (1.281%)  43 Rapa = 30.9 mg (1.03%) S No, 50 μL PEG 400 =2.9624 g (98.97%)  44 Rapa = 61 mg (1.96%) S Yes, 50 μL Ethanol, 100% =0.1860 g (6%) PEG 400 = 2.8588 g (92.04%)  45 Rapa = 90.7 mg (3.02%) SYes, 50 μL Ethanol, 100% = 0.2722 g (9.06%) PEG 400 = 2.6423 g (87.94%) 46 Rapa = 101.6 mg (4.997%) S EtOH = 331.6 mg (16.308%) PEG400 = 1600.1mg (78.695%)  47 Rapa = 120.9 g (3.189%) SP F68(Lutrol) = 42.4 mg(1.118%) Sterile Water = 3627.7 mg (95.692%)  48 Rapa = 100.1 g (1.999%)S EtOH = 305.1 mg (6.092%) PEG400 = 4602.9 mg (91.909%)  49 Rapa = 150.5mg (3.004%) SP Yes, 20 μL, 40 μL PEG400 = 4860.3 mg (96.996%)  50 Rapa =153.4 mg (3.055%) SP No, 20 μL F68(Pluronic) = 50.6 mg (1.008%) SterileWater = 4816.6 mg (95.937%)  51 Rapa = 116.6 mg (2.29%) S Yes, 30 μLEtOH = 306.6 mg (6.05%) PEG400 = 4647.5 mg (91.66%)  52 Rapa = 150.4 mg(2.994%) SP F68 Lutrol = 15.4 mg (0.306%) Sterile water = 4859.1 mg(96.7%)  53 Rapa = 306.5 mg (6.088%) SP PEG 400 = 4727.7 mg (93.912%) 54 Rapa = 309.3 mg (6.146%) SP PEG 400 = 4723.3 mg (93.854%)  55 Rapa =303.3 mg (6.061%) SP PEG 400 = 4700.6 mg (93.939%)  56 Rapa = 305.4 mg(6.088%) SP PEG 400 = 4711.0 mg (93.912%)  57 Rapa = 306.9 mg (6.098%)SP PEG 400 = 4725.5 mg (93.902%)  58 Rapa = 302.5 mg (6.021%) SP PEG 400= 4721.6 mg (93.979%)  59 Rapa = 304.5 mg (6.053%) SP PEG 400 = 4726.4mg (93.947%)  60 Dexamethasone = 251.4 mg (5.011%) SP PEG 400 = 4765.2mg (94.989%)  61 Dexamethasone = 252.4 mg (5%) SP PEG 400 = 4600 mg(92%) EtOH = 150 mg (3%)  62 Rapa = 32.2 mg (0.641%) S PEG 400 = 4677.9mg (93.096%) EtOH = 314.7 mg (6.263%)  63 Rapa = 32.3 mg (0.6%) S PEG400 = 5516.3 mg (93.1%) EtOH = 314.7 mg (6.263%)  64 Rapa = 54.4 mg(1.007%) S PEG 400 = 4638.9 mg (92.702%) EtOH = 314.8 mg (6.291%)  65Rapa = 50.8 mg (1.013%) S PEG 400 = 4963.2 mg (98.987%)  66 Rapa = 52.1mg (1.035%) S PEG 400 = 4868.6 mg (96.718%) EtOH = 113.1 mg (2.247%)  67Rapa = 50.5 mg (1.009%) S Yes, 20 μL PEG 400 = 4752.8 mg (94.953%) No,40 μL, 100 μL EtOH = 202.1 mg (4.038%)  68 Rapa = 101.8 mg (2.030%) SPEG 400 = 4712.4 mg (93.970%) EtOH = 200.6 mg (4.000%)  69 Rapa = 102.1mg (2.036%) S PEG 400 = 4605.5 mg (91.847%) EtOH = 306.7 mg (6.117%)  70Rapa = 101.6 mg (2.025%) S PEG 400 = 4510.6 mg (89.892%) EtOH = 405.6 mg(8.083%)  71 Rapa = 75.9 mg (3.019%) SP PEG 400 = 2438.4 mg (96.981%) 72 Rapa = 50.9 mg (2.034%) S PEG 400 = 2350.1 mg (93.914%) EtOH = 101.4mg (4.052%)  73 Rapa = 12.5 mg (0.620%) SP PEG 400 = 2004.8 mg (99.380%) 74 Rapa = 1.20949 g (2.0152%) S EtOH = 2.401 g (4.000%) PEG 400 =56.407 g (93.9848%)  75 Rapa = 16.0 mg g (0.795%) S No, 50 μL EtOH =80.0 mg (3.976%) PEG 400 = 1916.0 mg (95.2298%)  76 Rapa = 8.1 mg(0.400%) SP PEG 400 = 2014.5 mg (99.600%)  77 Rapa = 8.6 mg (0.428%) SPEG 400 = 2002.5 mg (99.572%)  78 Rapa = 8.2 mg (0.410%) S PEG 400 =1992.0 mg (99.590%)  79 Rapa = 8.7 mg (0.433%) S PEG 400 = 1998.8 mg(99.567%)  80 Rapa = 8.6 mg (0.427%) S PEG 400 = 2003.2 mg (99.573%)  81Rapa = 8.6 mg (0.428%) S PEG 400 = 1999.3 mg (99.572%)  82 Rapa = 9.0 mg(0.448%) S PEG 400 = 2000.8 mg (99.552%)  83 Rapa = 8.0 mg (0.397%) SPEG 400 = 2008.8 mg (99.603%)  84 Rapa = 8.5 mg (0.422%) S PEG 400 =2006.8 mg (99.578%)  85 Rapa = 8.0 mg (0.399%) S PEG 400 = 1998.2 mg(99.601%)  86 Rapa = 8.5 mg (0.422%) S PEG 400 = 2004.3 mg (99.578%)  87Rapa = 8.6 mg (0.428%) S PEG 400 = 2002.5 mg (99.572%)  88 Rapa = 0.7 g(1.983%) S EtOH = 1.4 g (3.966%) PEG 400 = 33.2 g (94.051%)  89 Rapa = 0g (0%) S EtOH = 0.574 g (1.995%) PEG 400 = 28.2 g (98.005%)  90 Rapa =1.95 g (1.950%) S EtOH = 4.05 g (4.050%) PEG 400 = 94.00 g (94000.%)  91Rapa = 0.0107 g (0.534%) S No, 80 μL EtOH = 0.0805 g (4.019%) PEG 400 =1.912 g (95.447%)  92 Rapa = 0.0081 g (0.403%) S No, 100 μL EtOH =0.0804 g (4.003%) PEG 400 = 1.920 g (95.594%)  93 Rapa = 1.992 g (2%) SEtOH = 3.9419 (4%) PEG 400 = 93.95 g (94%)  94 Rapa = 0.405 g (0.4%) SEtOH = 4.24 g (4%) PEG 400 = 95.6 (95.6%)  95 PEG 400 = 96 g (96%) SEtOH = 3.9027 (4%)  96 Rapa = 0.4020 g (0.402%) S EtOH = 3.970 g(3.971%) PEG 400 = 95.600 g (95.627%)  97 Rapa = 2.000 g (1.990%) S EtOH= 4.000 g (3.980%) PEG 400 = 94.500 g (94.030%)  98 PEG 400 = 96 g (96%)S EtOH =3.92 g (4%)  99 Rapa = 0.4036 g (0.4%) S No, 100 μL EtOH =3.9054 g (4%) PEG 400 = 95.6 (95.6%) 100 Rapa = 2.0025 g (2%) S Yes, 1μL, 3 μL, EtOH = 3.98 g (4%) 20 μL, 40 μL PEG 400 = 94.00 g (94%) 101Rapa = 9.5 mg (0.472%) S EtOH = 90.3 mg (4.485%) PEG 600 = 1913.5 mg(95.043%) 102 Rapa = 44.6 mg (2.21%) S EtOH = 86.1.0 mg (4.26%) PEG 600= 1891.1 mg (93.53%) 103 Rapa = 1.97 g (2%) S EtOH = 4.10 g (4%) PEG 400= 94.15 g (94%) 104 Rapa = 1.95 g (2%) S EtOH = 4.00 g (4%) PEG 400 =94.0 g ( 94%) 105 Rapa = 8.00 g (2%) S PEG 400 = 376.0 g EtOH = 16.0 g(4%) 106 Rapa = 6.00 g (2%) S PEG 400 = 282.0 g (94%) EtOH = 12.0 g (4%)107 Rapa = 8.9 mg (0.4434%) S EtOH = 80.3 mg (4.0006%) PEG 300 = 1918.0mg (95.556%) 108 Rapa = 40.8 mg (2.00886%) S EtOH = 110.0 mg (5.41605%)PEG 300 = 1880.2 mg (92.57509%) 109 Rapa = 9.9 mg (0.488%) S EtOH = 86.7mg (4.277%) PEG 400/300(50/50) = 1930.3 mg (95.235%) 110 Dexamethasone =142.5 mg SP 0.3305 μm Yes, 30 μL (4.994%) PEG 400 = 2710.7 mg (95.006%)111 Dexamethasone = 134.3 mg SP >10 μm (4.891%) PEG 400 = 2611.4 mg(95.109%) 112 Triamcinolone = 139.2 mg (5.087%) SP 3.98 μm Yes, 30 μLPEG 400 = 2597.4 mg (94.913%) 113 Triamcinolone = 135.3 mg (5.089%)SP >10 μm PEG 400 = 2523.5 mg (94.911%) 114 EtOH = 206.4 mg (4.121%) SNo, 30 μL PEG 400 = 4801.6 mg (95.879%) 115 Rapa = 43.0 mg (2.144%) SP61.4390 μm PEG 400 = 1962.3 mg (97.8567%) 116 Rapa = 40.0 mg (2.001%) SP3.7128 μm PEG 400 = 1959.1 mg (97.999%) 117 Rapa = 42.9 mg (2.142%) SP2.7313 μm PEG 400 = 1959.7 mg (97.858%) 118 Rapa = 100.8 mg (2.013%) SP4.1063 μm PEG 400 = 4906.0 mg (97.987%) 119 Rapa = 20.9 mg (0.42%) SEtOH = 209.1 mg (4.17%) PEG 400 = 4784.9 mg (95.41%) 120 Rapa = 20.6 mg(0.41%) S EtOH = 211.5 mg (4.22%) Benz. Chl = 19.1 mg (0.38%) PEG 400 =4762.0 mg (94.99%) 121 Rapa = 20.1 mg (0.40%) S EtOH = 211.5 mg (4.22%)Benz. Chl = 2.3 mg (0.05%) PEG 400 = 4782.3 mg (95.34%) 122 Rapa = 8.0 g(2%) S EtOH = 16.0 g (4%) PEG 400 = 376.0 g (94%) 123 Rapa = 351.3 mg(2.006%) S EtOH = 2353.1 mg (4.093%) PEG 400 = 16448.2 mg (93.901%) 124Rapa = 2.2035 g (2%) S EtOH = 4.45 g (4%) PEG400 = 103.7 g (94%( 125Rapa = 515.5 mg (2.021%) SP 18.1453 μm PEG 400 = 24,993.8 mg (97.979%)126 Rapa = 0.3 g (2%) S EtOH = 0.6 g (4%) PEG 400 = 14.1 g (94%) BHT =0.0002 (0.002%) 127 Rapa = 0.3 g (2%) S EtOH = 0.6 g (4%) PEG 400 = 14.1g (94%) BHT = 0.00037 (0.004%) 128 Rapa = 0.3 g (2%) S EtOH = 0.6 g (4%)PEG 400 = 14.1 g (94%) BHT = 0.0081 (0.05%) 129 Rapa = 243.2 mg (1.869%)S EtOH = 4.88.4 mg (3.753%) PEG400 = 12283.3 mg (94.378%) 130 Rapa =0.404 g (2%) S EtOH = 0.8 g (4%) PEG 400 = 18.8 g (94%) BHT = 0.00051(0.002%) 131 Rapa = 0.6024 g (2%) S EtOH = 1.2 g (4%) PEG 400 = 28.25 g(94%) 132 Rapa = 2.001 g (2%) S EtOH = 4.05 g (4%) PEG 400 = 94.45 g(94%) 133 Rapa = 0.5155 g (2.057%) S EtOH = 1.0198 g (4.070%) PEG 400 =23.5225 g (93.873%) 134 PEG 400 = 9.6 g (96%) S EtOH = 0.4 g (4%) 135Rapa = 0.610 g (2%) S EtOH = 1.2 g (4%) PEG 400 = 28.2 g (94%) 136 Rapa= 24.6 mg (1.193%) S EtOH = 91.1 mg (4.418%) Tyloxapol = 219.6 mg(10.649%) BSS = 1726.8 mg (83.740%) 137 Rapa = 100.0 mg (1.993%) SP PEG400 = 4916.9 mg (98.007%) 138 Rapa = 201.6 mg (4.005%) SP PEG 400 =4831.5 mg (95.995%) 139 Rapa = 102.4 mg (2.036%) S EtOH = 209.0 mg(4.154%) PEG 400 = 4719.3 mg (93.810%) 140 Rapa = 10.3 mg (0.205%) SYes, 10 μL EtOH = 27.4 mg (0.544%) PEG 400 = 4995.8 mg (99.251%) 141Rapa = 10.6 mg (0.211%) S No, 10 μL EtOH = 208.4 mg (4.150%) PEG 400 =4802.3 mg (95.639%) 142 Rapa = 31.5 mg (0.628%) S Yes, 10 μL EtOH = 67.1mg (1.337%) PEG 400 = 4918.9 mg (98.035%) 143 Rapa = 30.8 mg (0.613%) SNo, 10 μL, 100 μL EtOH = 204.5 mg (4.073%) PEG 400 = 4786.1 mg (95.314%)144 Rapa = 103.5 mg (2.057%) S Yes, 10 μL EtOH = 207.1 mg (4.116%) PEG400 = 4720.8 mg (93.827%) 145 Rapa = 283.0 mg (2.020%) S EtOH = 566.1 mg(4.041%) PEG 400 = 13,160.8 mg (93.939%) 146 Rapa = 280.1 mg (1.998%) SEtOH = 565.2 mg (4.033%) PEG 400 = 13,171.7 mg (93.969%) 147 Rapa =201.6 mg (3.000%) SP PEG 400 = 6518.8 mg (97.000%) 148 Rapa = 31.9 mg(1.019%) S Benzyl Alcohol = 1021.9 mg (20.070%) Sesame Oil = 4017.9 mg(78.911%) 149 Rapa = 51.5 mg (1.03%) S Benzyl Alcohol = 259.9 mg (5.19%)Sesame Oil = 4694.3 mg (93.78%) 150 Rapa = 5.96 g (2%) S EtOH = 12.0 g(4%) PEG 400 = 282.0 g (94%) 151 Rapa = 54.5 mg (1.07%) S Benzyl Alcohol= 1014.3 mg (19.95%) Olive Oil = 4014.8 mg (78.98%) 152 Rapa = 0 mg(0.00%) S Benzyl Alcohol = 269.4 mg (5.421%) Tyloxapol = 608.2 mg(12.238%) Sesame Oil = 4092.2 mg (82.341%) 153 Rapa = 76.3 mg (1.75%) SBenzyl Alcohol = 307.0 mg (7.06%) Tyloxapol = 607.8 mg (13.97%) SesameOil = 3000.5 mg (68.97%) Span 80 = 63.1 mg (1.45%) EtOH = 295.5 mg(6.79%) 154 Form. #150 = 200 g (99.998) S BHT = 0.004 g (0.002%) 155Rapa = 51.0 mg (0.87%) S EtOH = 642.3 mg (10.93%) Benzyl Alcohol = 431.8mg (7.34%) Sesame Oil = 4753.7 mg (80.86%) 156 Rapa = 51.4 mg (1.03%) SBenzyl Alcohol = 518.4 mg (10.34%) Olive Oil = 4444.7 mg (88.64%) 157Rapa = 8.1 g (2%) S EtOH = 16.0 g (4%) PEG 400 = 376.0 g (94%) 158 Form.#157 = 225.00 g (99.998%) S BHT = 0.0045 g (0.002%) 159 Rapa = 8.1 g(2%) S EtOH = 16.0 g (4%) PEG 400 = 376 g (94%) 160 Form. #159 = 112.0 g(99.998%) S BHT = 0.00224 g (0.002%) 161 Form. #159 = 112.0 g (99.998%)S BHT = 0.0019 g (0.002%) 162 Rapa = 55.4 mg (1.10%) S EtOH = 112.7 mg(2.25%) Benzyl Alcohol = 157.8 mg (3.15%) Cotton Seed Oil = 4688.0 mg(93.50%) 163 Rapa = 5.005 g (1%) S EtOH = 10.0 g (2%) PEG 400 = 485.5 g(97%) 164 PEG 400 = 9.82 g (98%) S EtOH = 0.235 g (2%) 165 Form. #163 =100.25 g (99.998%) S BHT = 0.0026 g (0.002%) 166 Rapa = 203.1 mg(2.025%) SP 2.8651 μm F68 = 30.3 mg (0.303%) Sterile Water = 9792.6 mg(97.672%) 167 Rapa = 201.4 mg (2.0005%) SP 1.0984 μm Tween 20 = 43.9 mg(0.436%) Sterile Water = 9822.8 mg (97.564%) 168 EtOH = 0.8301 g(4.144%) S PEG 400 = 19.2014 g (95.856%) 169 Form. #168 = 300 μl S 170Form. #168 = 250 μl S Form. #154 = 50 μl 171 Form. #168 = 200 μl S Form.#154 = 100 μl 172 Form. #168 = 150 μl S Form. #154 = 150 μl 173 Form.#154 = 300 μl S 174 Rapa = 102.2 mg (2.041%) SP 0.4165 μm F68 = 16.0 mg(0.32%) Sterile Water = 4889.0 mg (97.639%) 175 Rapa = 101.1 mg (2.010%)SP 0.5294 μm Tween 20 = 27.7 mg (0.551%) Sterile Water = 4901.0 mg(97.439%) 176 BSS+ = 0 μl S Sterile Water = 0 μl Form. #154 = 1000 μl177 BSS+ = 200 μl SP Sterile Water = 0 μl Form. #154 = 800 μl 178 BSS+ =400 μl SP Form. #154 = 600 μl 179 BSS+ = 500 μl SP Form. #154 = 500 μl180 BSS+ = 600 μl SP Form. #154 = 400 μl 181 BSS+ = 800 μl SP Form. #154= 200 μl 182 Sterile Water = 200 μl SP Form. #154 = 800 μl 183 SterileWater = 400 μl SP Form. #154 = 600 μl 184 Sterile Water = 500 μl SPForm. #154 = 500 μl 185 Sterile Water = 600 μl SP Form. #154 = 400 μl186 Sterile Water = 800 μl SP Form. #154 = 200 μl 187 BSS+ = 2536.9 mg(49.98%) SP 60.2075 μm Form. #154 = 2538.7 mg (50.02%) 188 Sterile Water= 2515.6 mg (49.84%) SP 617.5157 μm Form. #154 = 2532.2 mg (50.16%) 189F68 = 12.6 mg (0.25%) SP 70.6089 μm Sterile Water = 2524.7 mg (49.79%)Form. #154 = 2533.1 mg (49.96%) 190 Rapa = 2.0225 g (2%) S EtOH = 3.65 g(4%) PEG 400 = 94.0 g ( 94%) BHT = 0.002 g (0.002%) 191 F68 = 12.1 mg SPSterile Water = 2558.9 mg Form. #154 = 2556.4 mg 192 F68 = 19.8 mg SPSterile Water = 2564.1 mg Form. #154 = 25557.5 mg 193 F68 = 25.3 mg SPSterile Water = 2575.1 mg Form. #154 = 2572.9 mg 194 F68 = 32.4 mg SPSterile Water = 2572.1 mg Form. #154 = 2562.1 mg 195 F68 = 38.3 mg SPSterile Water = 2563.2 mg Form. #154 = 2573.5 mg 196 F68 = 43.6 mg SPSterile Water = 2541.1 mg Form. #154 = 2556.0 mg 197 F68 = 51.2 mg SPSterile Water = 2594.5 mg Form. #154 = 2594.1 mg 198 PEG 400 = 1920 g(96%) S EtOH = 80 g (4%) 199 Form. #168 = 1000 μl S 200 Form. #168 = 200μl S Form. #154 = 800 μl 201 Form. #168 = 400 μl S Form. #154 = 600 μl202 Form. #168 = 500 μl S Form. #154 = 500 μl 203 Form. #168 = 600 μl SForm. #154 = 400 μl 204 Form. #168 = 800 μl S Form. #154 = 200 μl 205PEG 400 = 200 μl S Form. #154 = 800 μl 206 PEG 400 = 400 μl S Form. #154= 600 μl 207 PEG 400 = 500 μl S Form. #154 = 500 μl 208 PEG 400 = 600 μlS Form. #154 = 400 μl 209 PEG 400 = 800 μl S Form. #154 = 200 μl 210Phosal 50PG = 6735.0 mg S (99.002%) Tween 80 = 67.9 mg (0.998%) 211 Rapa= 2.0047 g (2%) S EtOH = 4.00 g (4%) PEG 400 = 94.05 g (94%) 212 Phosal50PG = 20.0662 g S (98.999%) Tween 80 = 0.2029 g (1.001%) 213 Form. #154= 100 μl S Form. #168 = 900 μl 214 Form. #154 = 100 μl S Form. #168 =900 μl 215 Form. #154 = 100 μl S Form. #168 = 900 μl 216 Form. #154 =100 μl S PEG 400 = 900 μl 217 Form. #154 = 100 μl S PEG 400 = 900 μl 218Form. #154 = 100 μl S PEG 400 = 900 μl 219 Form. #154 = 100 μl SP BSS+ =900 μl 220 Form. #154 = 100 μl SP BSS+ = 900 μl 221 Form. #154 = 100 μlSP BSS+ = 900 μl 222 Form. #154 = 1000 μl S 223 Form. #154 = 1000 μl S224 Form. #154 = 100 μl S Form. #168 = 900 μl 225 Form. #154 = 100 μl SForm. #168 = 900 μl 226 Form. #154 = 100 μl S Form. #168 = 900 μl 227Form. #154 = 100 μl S PEG 400 = 900 μl 228 Form. #154 = 100 μl S PEG 400= 900 μl 229 Form. #154 = 100 μl S PEG 400 = 900 μl 230 Form. #154 = 100μl SP BSS+ = 900 μl 231 Form. #154 = 100 μl SP BSS+ = 900 μl 232 Form.#154 = 100 μl SP BSS+ = 900 μl 233 Form. #154 = 200 μl S Form. #168 =800 μl 234 Form. #154 = 200 μl S Form. #168 = 800 μl 235 Form. #154 =200 μl S Form. #168 = 800 μl 236 Form. #154 = 200 μl S Form. #168 = 800μl 237 Form. #154 = 200 μl S PEG 400 = 800 μl 238 Form. #154 = 200 μl SPEG 400 = 800 μl 239 Form. #154 = 200 μl SP BSS+ = 800 μl 240 Form. #154= 200 μl SP BSS+ = 800 μl 241 Form. #154 = 200 μl SP BSS+ = 800 μl 242Form. #154 = 100 μl S No, 10 μL Form. #168 = 900 μl 243 Form. #154 = 100μl S Yes, 10 μL PEG 400 = 900 μl 244 Form. #154 = 100 μl SP Yes, 10 μLBSS+ = 900 μl 245 Form. #154 = 100 μl SP BSS+/CMC(0.5%) = 900 μl 246Form. #154 = 400 μl S No, 10 μL Form. #168 = 900 μl 247 Form. #154 = 400μl S Yes, 10 μL PEG 400 = 900 μl 248 Form. #154 = 400 μl SP Yes, 10 μLBSS+ = 900 μl 249 Form. #154 = 400 μl SP BSS+/CMC(0.5%) = 900 μl 250Form. #154 = 100 μl SP BSS+/CMC(0.5%) = 900 μl 251 Form. #154 = 100 μlSP BSS+/CMC(0.5%) = 900 μl 252 Form. #154 = 100 μl SP BSS+/CMC(0.5%) =900 μl 253 Form. #154 = 200 μl SP BSS+/CMC(0.5%) = 800 μl 254 Form. #154= 200 μl SP BSS+/CMC(0.5%) = 800 μl 255 Form. #154 = 200 μl SPBSS+/CMC(0.5%) = 800 μl 256 Form. #154 = 400 μl SP BSS+/CMC(0.5%) = 900μl 257 Form. #154 = 400 μl SP BSS+/CMC(0.5%) = 900 μl 258 Form. #154 =400 μl SP BSS+/CMC(0.5%) = 900 μl 259 EtOH = 17.1 mg (0.57%) S PEG 400 =2997.3 mg (99.43%) 260 EtOH = 40.8 mg (1.35%) S PEG 400 = 2980.2 mg(98.65%) 261 EtOH = 47.1 mg (1.57%) S PEG 400 = 2950.1 mg (98.43%) 262Rapa = 2.0032 g (2%) S EtOH = 3.92 g (4%) PEG 400 = 94.00 g (94%) 263Triamcinolone acetomide = SP 80.8 mg (4.04%) PEG 400 = 1920.8 mg(95.96%) 264 NFF-0007 filled in glove box S 265 PEG 400 = 9.598 g (96%)S EtOH = 0.4052 (4%) 266 Triamcinolone acetomide = SP 42.2 mg (4.123%)PEG 400 = 981.3 mg (95.877%) 267 Phosal 50PG = 20.0783 g S (99.00835%)Tween 80 = 0.2011 g (0.99165%) 268 PEG 400 = 96.1 g (96%) S EtOH = 4.00g (4%) 269 Rapa = 0.4001 g (2%) S EtOH = 0.80 g (4%) PEG 400 = 18.8 g(94%) 270 Sterile Water = 9955.8 mg (99.27%) S CMC High visc. = 47.8 mg(0.48%) Tween 80 = 25.4 mg (0.25%) 271 Sterile Water = 9947.5 mg(99.00%) S CMC Medium visc. = 75 mg (0.75%) Tween 80 = 25.1 mg (0.25%)272 Rapa = 41 mg (2.01%) SP Form. #270 = 2000 mg (97.99%) 273 Rapa =40.2 mg (1.97%) SP MSF-03-172-07E = 2000 mg (98.03%) 274 NMP(Pharmasolve^( ® )) = 1280.5 mg S (65.89%) PLGA 75/25 = 662.9 mg(34.11%) 275 NMP (Pharmasolve^( ® )) = 1573.3 mg S (80.50%) PLGA 75/25 =381.0 mg (19.50%) 276 NMP (Pharmasolve^( ® )) = 1009.7 mg S Yes, 10 μL49.8%) PLGA 75/25 = 1001.6 mg (50.20%) 277 Sterile Water = 14934.0 mg(99.25%) S CMC Medium visc. = 112.4 mg (0.75%) 278 Propylene Glycol =1893.7 mg S Yes, 10 μL (93.85%) EtOH = 83.8 mg (4.16%) Rapa = 40.2 mg(1.99%) 279 Propylene Glycol = 1946.2 mg S Yes, 10 μL (95.68%) BenzylAlcohol = 47.1 mg (2.31%) Rapa = 40.8 mg (2.01%) 280 PEG 300 = 1894.1 mg(93.74%) S Yes, 10 μL EtOH = 40.1 mg (1.98%) Rapa = 86.4 mg (4.28%) 281PEG 300 = 1925.5 mg (95.88%) S Yes, 10 μL, 30 μL EtOH = 39.8 mg (1.98%)Rapa = 43.0 mg (2.14%) 282 Rapa = 100.6 mg (2.01%) SP Yes, 10 μL, 30 μLMSF-03-176-02 = 4910.8 mg (97.99%) 283 Rapa = 11.5 mg (0.57%) S PEG 300= 2012.5 mg (99.43%) 284 Rapa = 10.3 mg (0.51%) S PEG 400 = 2017.2 mg(99.49%) 285 Rapa = 9.8 mg (0.486%) S PEG 600 = 2005.9 mg (99.51%) 286Tacrolimus = 42.7 mg (2.11%) S EtOH = 46.0 mg (2.27%) PG = 1938.7 mg(95.62%) 287 Tacrolimus = 40.7 mg (2.01%) S EtOH = 43.0 mg (2.12%) PEG300 = 1942.1 mg (95.87%) 288 Tacrolimus = 40.3 mg (1.99%) S EtOH = 43.8mg (2.16%) PEG 400 = 1942.3 mg (95.85%) 289 Tacrolimus = 40.8 mg (2.03%)S EtOH = 44.5 mg (2.21%) PEG 600 = 1924.0 mg (95.76%) 290 Rapa = 61.0 mg(3.17%) S NMP = 1226.54 mg (63.80%) PLGA 75/25 = 634.96 mg (33.03%) 291Rapa = 100.2 mg (5.13%) S NMP = 1492.95 mg mg (76.37%) PLGA 75/25 =361.65 mg (18.50%) 292 Rapa = 62.9 mg (3.04%) S NMP = 1103.8 g mg(53.40%) PLGA 75/25 = 900.2 mg (43.56%) 293 Rapa = 62.4 mg (3.00%) S NMP= 1205.1 mg mg (58.11%) PLGA 75/25 = 806.4 mg (38.89%) 294 SterileWater + 1% CMC Med. = SP 4909.1 mg (97.99%) Rapa = 100.5 mg (2.01%) 295Sterile Water + 1% CMC high. = SP 4903.8 mg (97.96%) Rapa = 101.9 mg(2.04%) 296 Rapa = 40.5 mg (2.03%) S NMP = 1958.7 mg (97.97%) 297 Rapa =20.5 mg (2.0%) SP DMA = 41.4 mg (4.0%) PVP = 35.0 mg (3.4%) H2O = 934.7mg (90.6%) 298 Rapa = 10.6 mg (2.0%) S DMA = 10.6 mg (2.0%) PEG 400 =506.1 mg (96%) 299 Rapa = 5.2 mg (2.0%) SP 1% DMA in PEG 400 = 257.4 mg(98%) 300 Rapa = 20.0 mg (2.0%) S DMA = 7.8 mg (0.8%) PEG 400 = 974 mg(97.2%) 301 Rapa = 20.1 mg (1.3%) S DMA = 19.5 mg (1.3%) PEG 400 =1449.6 mg (97.3%) 302 Rapa = 20.0 mg (2.0%) SP PVP = 10.8 mg (1.1%) PEG400 = 994.5 mg (97.0%) 303 Rapa = 20.4 mg (2.0%) SP PVP = 24.5 mg (2.4%)PEG 400 = 990.7 mg (95.7%) 304 Rapa = 25.5 mg (2.4%) SP PVP = 51.9 mg(4.8%) PEG 400 = 1000.6 mg (92.8%) 305 Rapa = 22.5 mg (2.3%) S BA = 27.5mg (2.7%) PEG 400 = 950.7 mg (95.0%) 306 Rapa = 30.2 mg (2.3%) SP PVP =240.9 mg (18.6%) PEG 400 = 1021.2 mg (79.0%) 307 Rapa = 8.7 mg (3.1%) SP1% PVP in H2O = 273 mg (96.9%) 308 Rapa = 12.6 mg (2.53%) SP 5% PVP inH2O = 501.6 mg (97.5%) 309 Rapa = 20.3 mg (3.8%) SP 10% PVP in H2O =513.9 mg (96.2%) 310 Rapa = 100.5 mg (2.0%) S Yes, 10 μL DMA = 67.8 mg(1.4%) PEG 400 = 4838.3 mg (96.6%) 311 Rapa = 96.8 mg (1.9%) S Yes, 10μL BA = 157.5 mg (3.2%) PEG 400 = 4748.7 mg (94.9%) 312 Rapa = 105.8 mg(2.1%) S DMA = 5.63 mg (0.1%) PEG400 = 4888.9 mg (97.8%) 313 Rapa = 20.2mg (2.0%) SP PVP = 99.2 mg (9.9%) H2O = 882.3 mg (88.1%) 314 Rapa =100.3 mg (2.0%) SP PVP = 251.4 mg (5.0%) H2O = 4662.8 mg (93.0%) 315Rapa = 20.3 mg (2.0%) S DMA = 983.9 mg (98%) 316 Triamcinolone = 22.8 mg(2.0%) S Yes, 10 μL DMA = 12.0 mg (1.1%) PEG400 = 1104.5 mg (96.9%) 317Triamcinolone = 1.0 mg (0.1%) S EtOH = 49.30 mg (4.0%) PEG 400 = 1191.9mg (96.0%) 318 Triamcinolone = 18.7 mg (0.9%) S PEG 400 = 959.8 mg(99.1%) 319 Triamcinolone = 25.5 mg (1.3%) S EtOH = 83.0 mg (4.1%) PEG400 = 1905.6 mg (94.6%) 320 Dexamethasone = 20.4 mg (1.2%) S EtOH = 71.7mg (4.1%) PEG400 = 1737.6 mg (98.8%) 321 Dexamethasone = 27.5 mg (2.0%)S Yes, 10 μL DMA = 5.6 mg (0.4%) PEG 400 = 1347.3 mg (97.6%) 322 Rapa =9.1 mg (0.152%) E EtOH = 90.9 mg (1.514%) F127 = 262.8 mg (4.378%) Water= 1489.1 mg (24.804%) Sesame oil = 4151.5 mg (69.152%) 323 Rapa = 24.4mg (0.625%) E Phosal 50PG = 203.1 mg (5.201%) EtOH = 166.8 mg (4.272%)Labrafac CC = 1502.8 mg (38.486%) Sesame oil = 2007.7 mg (51.416%) 324Form. #174 with 2 mm beads SP 0.4929 μm 325 Form. #175 with 2 mm beadsSP 0.4804 μm

TABLE 2 Aqueous Humor Rapa Concentration Injection of 2% Mean RapaStandard Rapa-PEG-EtOH concentration deviation Solution (ng/mL) (ng/mL) 1.0 μL intravitreal 0.438 (1 day after injection) 0.141  3.0 μLintravitreal 0.355 (1 day after injection) 0.234  3.0 μL sub-conj 0.338(1 day after injection) 0.122  5.0 μL into anterior chamber   0.167 (14days after injection) 0.183 10.0 μL into anterior chamber   0.004 (14days after injection) 0.006

1. A method for treating age-related macular degeneration (AMD) in ahuman subject, the method comprising administering to the human subjectby intravitreal injection a volume of a liquid formulation comprising aneffective amount of a therapeutic agent to treat AMD in the humansubject for at least one month, wherein the liquid formulation comprisesbetween 0.5% (w/w) to 6.0% (w/w) of the therapeutic agent, between 80%(w/w) to 99% (w/w) of polyethylene glycol, and ethanol, the therapeuticagent is rapamycin or a pharmaceutical acceptable salt thereof, the AMDis selected from the group consisting of dry form of AMD, wet form ofthe AMD and transition from the dry form of AMD to the wet form of AMD,and the liquid formulation is a liquid solution that forms anon-dispersed mass when injected into the vitreous.
 2. The method ofclaim 1, wherein the AMD is the dry form of AMD.
 3. The method of claim1, wherein the at least one month is at least two months.
 4. The methodof claim 1, wherein the at least one month is at least three months. 5.The method of claim 1, wherein the polyethylene glycol and the ethanoldiffuse out of the non-dispersed mass upon said injection leaving aprecipitate of the therapeutic agent.
 6. The method of claim 1, whereinthe non-dispersed mass does not form a gel or gel-like substance uponinjection.
 7. The method of claim 1, wherein the liquid formulation hasa viscosity of between 40 and 120 centipoise.
 8. The method of claim 1,wherein the liquid formulation as a viscosity of between 60 and 80centipoise.
 9. The method of claim 1, wherein the therapeutic agent israpamycin.
 10. The method of claim 2, wherein the AMD is the wet form ofAMD.
 11. The method of claim 9, wherein the liquid formulation comprisesabout 2% (w/w) rapamycin, about 94% (w/w) PEG 400, and about 4% (w/w)ethanol.
 12. The method of claim 9, wherein the volume of liquidformulation contains between 20 μg to 2 mg rapamycin.
 13. The method ofclaim 1, wherein the therapeutic agent comprises between 0.628% to5.233% of the total weight of the liquid formulation.
 14. The method ofclaim 1, wherein the therapeutic agent comprises between 1% to 5% of thetotal weight of the liquid formulation.
 15. The method of claim 1,wherein the polyethylene glycol is PEG 300 or PEG
 400. 16. The method ofclaim 1, wherein the polyethylene glycol is PEG
 400. 17. The method ofclaim 1, wherein the at least one month is from one to three months. 18.The method of claim 1, wherein the AMD is the transition from the dryform of AMD to the wet form of AMD.
 19. A method of administering aliquid formulation, the method comprising: administering to a humansubject by intravitreal injection a volume of a liquid formulationcomprising between 0.5% (w/w) to 6.0% (w/w) of a therapeutic agent,between 80% (w/w) to 99% (w/w) of polyethylene glycol, and ethanol,wherein the therapeutic agent is rapamycin or a pharmaceuticalacceptable salt thereof, the human subject has a form of age-relatedmacular degeneration (AMD) selected from the group consisting of dryform of AMD, wet form of the AMD and transition from the dry form of AMDto the wet form of AMD, and the liquid formulation is a liquid solutionthat forms a non-dispersed mass when injected into the vitreous.
 20. Themethod of claim 19, wherein the administering is done once every two tothree months.
 21. The method of claim 19, wherein the therapeutic agentis rapamycin.